UNIVERSITY  OF  CALIFORNIA   PUBLICATIONS 

IN 

AGRICULTURAL    SCIENCES 

Vol.  4,  No.  1,  pp.  1-66,  pis.  1-2,  15  text-figures  December  31,  1918 


THE  FERMENTATION  ORGANISMS  OF 
CALIFORNIA  GRAPES1 


BY 

W.  V.   CEUESS 


CONTENTS 

PAGE 

Introduction 3 

I.     General  discussion  of  grape  organisms 3 

Molds 4 

Penicillium 4 

Aspergillus  niger 4 

Oidium 4 

Borytis  cinerea 6 

Mucor : 6 

Monilia 6 

True  Yeasts 6 

S.  ellipsoideus 6 

S.  cerevisiae 8 

S.  malei 8 

S.  pastorianus 8 

S.  anomalus 8 

S.  ludiwigii 9 

S.  marxianus 9 

Psuedo  Yeasts 9 

Apiculatus 9 

Mycoderma 9 

Torula 10 

1 1 .     Properties  of  molds  and  bacteria  from  California  grapes 10 

Sources  of  cultures 10 

Molds 12 

Penicillium 12 

Olive  color  penicillium  species 12 

Aspergillus 12 

Mucor  14 

Dematium 14 

Monilia 14 

Green  molds  (unidentified) 14 

Bacteria 14 


i  The  writer  wishes  to  thank  Professor  F.  T.  Bioletti  for  his  helpful  sug- 
gestions for  carrying  out  these  investigations  and  preparation  of  the  manuscript. 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

PAGE 

III.  Characteristics  of  yeasts  from  California  grapes 16 

Methods  of  study 16 

Morphology 16 

Fermentation  tests 16 

Yeasts  studied 18 

Apiculatus  yeasts 18 

Organism  35  (Apiculatus)  morphology 18 

Organism  72  (Apiculatus)  morphology 19 

Organisms  35  and  37,  rates  of  fermentation 19 

Mycoderma  forms 22 

Organism  65,  morphology 22 

Organism  68,  morphology 23 

Organism  70,  morphology 23 

Organism  71,  morphology 24 

Organism  73,  morphology 24 

Organism  76,  morphology 25 

Organism  78,  morphology 25 

Fermentation  tests  of  Mycoderma  forms 26 

Torula  forms 30 

Organism  37,  morphology 30 

Organism  77,  morphology 31 

Fermentation  tests  of  torula  yeast 32 

S.  Pastorianus  and  Willia  yeast 34 

Organism  36,  (S.  Pastorianus)  morphology 34 

Organism  69  (Willia  species)  morphology 35 

Fermentation  tests  of  S.  Pastorianus  and  Willia 35 

True  wine  yeasts,  S.  Ellipsoideus 38 

Organism  64,  morphology 38 

Organism  66,  morphology 39 

Organism  67,  morphology 39 

Organism  74,  morphology 40 

Organism  75,  morphology 40 

Organism  79,  morphology 41 

Fermentation  records  of  true  wine  yeasts  at  33°  C 41 

Attenuation  and  alcohol  formation  at  33°  C 44 

Comparison  of  fermentations  at  33°C  and  24°C  (all  varieties)....  46 

IV.  Influence  of  locality  on  the  character  of  the  micro-organisms  on  grapes..  50 

Methods  of  taking  samples 51 

Methods  of  counting  living  cells  on  grapes 51 

Results  of  countings 52 

Micro-organisms  on  grapes  from  Davis 52 

Micro-organisms  on  grapes  from  El  Centro 52 

Micro-organisms  on  grapes  from  Fresno 52 

Micro-organisms  on  grapes  from  Martinez 53 

Micro-organisms  on  grapes  from  Ripon 53 

Yeasts  from  the  Tulare  experiment  station 53 

Discussion  of  results 53 

V.     Influence  of  the  stage,  of  ripeness  on  the  character  of  the  micro-organisms 

on  grapes 54 

Sampling 54 

Results 55 


1918]                Cruess:   Fermentation  Organisms  of  California  Grapes  3 

PAGE 

VI.     Changes  in  the  numbers  and  character  of  the  micro-organisms  on  grapes 

during  shipment  from  vineyard  to  cellar 56 

VII.     Character  and  number  of  micro-organisms  on  grapes  as  received  at  the 

winery  during  the  seasons  of  1911  and  1912 57 

VIII.     Experiments  upon  the  control  of  micro-organisms  on  grapes  for  wine 

making 59 

Before  shipment 59 

After  arrival  at  cellar 62 

Summary  of  parts  II  -  VIII  (inclusive) 63 


INTRODUCTION 

The  manufacture  of  wine,  grape-juice,  and  raisins  and  the  shipping 
of  grapes  depend  very  largely  upon  the  control  of  micro-organisms, 
particularly  of  those  occurring  naturally  upon  the  fruit.  This  eco- 
nomic fact  and  the  scientific  interest  of  the  subject  led  to  a  study  of 
the  micro-organisms  occurring  on  the  grapes  of  California.  The 
investigation  was  qualitative  and  quantitative,  covering  the  effect  on 
type  and  number  of  micro-organisms  of  (a)  locality,  (6)  degree  of 
ripeness,  (c)  shipment  from  vineyard  to  winery.  It  included  studies 
of  (d)  the  micro-organisms  normally  found  on  grapes  as  received  at 
the  winery,  (e)  their  control  in  fermentation,  and  (/)  their  morpho- 
logical and  physiological  characteristics. 


I.  GENERAL   DISCUSSION    OF    GRAPE    MICRO-ORGANISMS 

Several  groups  of  micro-organisms  are  normally  found  on  grapes. 
These  may  be  classified  under  the  general  terms :  (1)  Budding  fungi, 
including  molds,  true  yeasts  which  form  spores,  and  pseudo-yeasts, 
which  do  not  form  spores;  and  (2)  fission  fungi,  including  bacteria 
(non-motile  rods),  bacilli  (motile  rods),  the  various  forms  of  Coc- 
caceae,  and  the  fission  yeasts. 

Representatives  of  all  the  above  groups  except  the  bacilli,  Coc- 
caceae,  and  fission  yeasts  were  found  in  California  grapes.  Most 
attention  has  been  given  to  the  fermentation  organisms:  i.e.,  yeasts. 
The  molds  and  bacteria  have  in  most  cases  been  merely  listed. 

The  more  important  forms  of  organisms  occurring  on  grapes  are 
the  following: 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


Molds 

Penicillium. — This  group  includes  a  large  number  of  varieties, 
which  are  the  most  widely  distributed  and  common  of  all  the  molds. 
They  are  characterized  by  the  method  of  formation  and  grouping  of 
conidia  shown  in  a  typical  manner  in  figure  1. 

The  most  common  mold  of  this  group  met  with  on  grapes  is  Peni- 
cillium glaucum  or  Penicillium  expansum.  It  is  the  common  green 
mold  and  is  the  cause  of  moldy  flavors  in  grapes,  barrels,  etc. 

During  the  first  stages  of  growth  it  appears  as  a  cottony,  white 
mass  of  mycelial  threads.  These  white  threads  soon  develop  fructifi- 
cations bearing  large  numbers  of  conidia  that  are  green  in  young 
cultures  and  brown  in  old.  These  give  a  powdery  appearance  to  the 
culture.  The  individual  conidiophores  are  branching  and  present  a 
broomlike  appearance  under  the  microscope. 

It  does  not  carry  on  alcoholic  fermentation,  but  may  destroy  sugar 
by  oxidation  to  C02  and  H20  or  the  formation  of  penicillic  acid. 

Aspergillus  niger. — This  is  a  black  mold  of  very  common  occur- 
rence on  California  grapes  (see  fig.  1).  On  grapes  and  must  it  forms 
a  white  mycelium  from  which  spring  short  rods  bearing  aggregations 
of  black  cells.  These  conidiophores  are  not  branching,  thus  differing 
from  penicillium.  These  groups  of  cells  are  easily  discernible  with 
the  unaided  eye.  On  the  grapes  of  some  districts  the  spores  or  conidia 
may  be  broken  away  from  the  main  growth  of  the  mold  during  pick- 
ing and  rise  as  a  black  dust.  At  present  it  is  not  held  to  be  very 
harmful  in  wine  making. 

Oidium  or  Powdery  Mildew  of  the  Vine. — This  fungus  is  of  more 
concern  to  the  grape  grower  than  it  is  to  the  wine  maker  and  will  be 
found  fully  described  in  Bulletin  1862  of  the  University  of  California 
Agricultural  Experiment  Station  (see  fig.  1).  This  mold  may  pre- 
vent the  grapes  reaching  maturity  or  may  cause  them  to  crack  and 
thus  to  be  liable  to  attack  by  penicillium.  In  both  cases,  the  grapes 
affected  may  become  unfit  for  wine  making.  The  microscopical  appear- 
ance of  the  summer  form  of  the  powdery  mildew  is  shown  in  figure  1. 
It  occurs  most  commonly  as  a  downy  white  growth  on  the  leaves,  canes, 
and  grapes  during  moist  or  foggy  weather  and  is  most  prevalent  dur- 
ing early  summer.  It  forms  winter  spores  or  perithecia  on  the  canes, 
in  which  form  the  organism  lives  through  the  winter. 


zBioletti,  V.  T.    (Oidium  or  Powdery  Mildew  of  the  Vine),  Univ.  Calif.  Exp. 
Si;,..  Bull.  ISO,  pp.  317-327,  1907. 


1918]  Cruess:    Fermentation  Organisms  of  California  Grapes 


/  FZNICILLIUM  k80O       Z.ftSPEiRGt  LL  1/5*500 


3-BOTFtVTUSxdOO  -f  MUCOR*  /SO- 


SOIDI UM x dOO    €>E>FKO VV/V  MOL 0*800 


Figure    1 
Molds  from  California  grapes: 

1.  Penicillium  variety  from  California  grapes,  magnified  800  diameters. 

2.  Aspergillus  variety  from  California  grapes;    X   800. 

3.  Botrytis  cinerea  from   California  grapes;    X    800. 

4.  Mucor  variety  from  California  grapes;    X    150. 

5.  Oidium  or  Powdery  Mildew  from  California  vine,  summer  form;    X   800. 

6.  Brown  mold,  parasitic  fungus  from  California  grapes;  X  800.     Species 

and  variety  not   determined. 


6  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Botrytis  cinerea. — This  mold  is  a  parasite  or  facultative  sapro- 
phyte occurring  on  grapes  in  moist  climates  (see  fig.  1).  It  rarely 
develops  on  California  grapes  during  the  wine  making  season  but  is 
often  found  on  grapes  left  on  the  vine  during  the  winter.  It  appears 
as  a  gray,  matted  growth  on  the  surface  of  the  grapes.  The  indi- 
vidual fructifications  may  be  seen  with  the  naked  eye  on  short  upright 
conidiophores.  Under  the  microscope,  the  conidia  may  be  seen  in 
grapelike  clusters. 

The  mold  did  not  grow  readily  in  culture  media,  but  will  develop 
profusely  on  infected  grapes  in  a  moist  jar. 

In  Europe,  the  Botrytis  is  considered  beneficial  in  the  Sauternes 
district,  because  it  causes  a  concentration  of  the  sugar  content  of  the 
grapes  by  favoring  evaporation  of  the  moisture.  It  produces  also  an 
oxydase  that  acts  vigorously  upon  the  color  of  the  grapes  or  wine. 

Mucor. — Mucor  mold  will  nearly  always  be  found  in  an  examina- 
tion of  California  grapes  (see  fig.  1).  Since  it  develops  very  slowly 
in  comparison  with  the  commoner  molds  and  yeasts  found  on  grapes 
it  is  not  probable  that  it  does  very  much  harm  in  wine  making. 

In  pure  cultures,  it  produces  a  gray  filamentous  mass  of  mycelial 
threads,  from  which  upright  rods  are  given  off,  bearing  at  their  upper- 
most ends  spherical  sporangia  filled  with  large  numbers  of  spores. 
These  sporangia  are  easily  seen  with  the  unaided  eye.  Most  of  the 
mucor  molds  are  capable  of  forming  yeastlike  cells  and  carrying  on  a 
feeble  alcoholic  fermentation  in  sugary  liquids. 

Monilia. — The  monilia  molds  and  especially  Monilia  Candida  occur 
very  commonly  on  fruits  of  all  kinds  (see  fig.  2).  Most  of  the  grapes 
examined  in  1912  bore  considerable  numbers  of  the  cells  of  this 
organism. 

In  young  cultures,  it  forms  colorless  yeastlike  cells  and  gives  a 
feeble  fermentation.  The  fermentation  is  followed  by  a  moldlike 
growth  on  the  surface  of  the  grape  must,  etc.,  which  in  old  cultures 
becomes  olive  green  in  spots. 


True  Yeasts 

Culture  Yeasts. —  (1)  Saccharomyces  ellipsoideus  (True  Wine 
Yeast).  In  general,  the  ellipsoideus  yeasts  are  characterized  by  rapid 
growth  in  grape  must  with  the  production  of  a  strong  fermentation, 
yielding  10%  to  16%  of  alcohol.  They  differ  from  the  beer  yeasts, 
8.  cerevisiae,  principally  in  their  higher  alcohol-forming  power  and 


1918] 


Cruess:   Fermentation  Organisms  of  California  Grapes 


ill  the  flavor  of  the  fermented  liquids,  the  wine  yeast  giving  a  vinous 
flavor  to  fermented  liquids  and  the  beer  yeast  a  beer  flavor.  In  grape 
must,  the  S.  ellipsoideus  forms  a  cloudy  growth  and  a  pasty  or  gran- 
ular sediment  in  the  bottom  of  the  container  during  the  main  fer- 
mentation.   After  fermentation  is  complete  the  suspended  cells  settle 


I.DEMflTIUMxSOa      %  MONILW   x  800 


3.  VI  A/e6/7/=f  BftCT£/?//9x/OCO  ^  TOURNC  BnCTOf/ffx/OOO. 


Figure    2 
Molds  and  yeasts  from  California  grapes: 

1.  Dematium  variety  from  California  grapes;    X    800. 

2.  Monilia  variety  from  California  grapes;    X   800. 

3.  Vinegar  bacteria  from  California  grapes;    X    1000. 

4.  Tourne  bacteria  from   California  wine;    X    1000. 

out,  giving  a  clear  liquid  and  compact  sediment.  Under  suitable  con- 
ditions spores  are  formed.  The  usual  shape  of  the  cells  is  shortly 
ellipsoidal,  although  this  will  vary  from  spherical  to  elongate,  with 
the  different  varieties.  The  shape  of  the  spores  is  spherical.  The 
appearance  of  several  specimens  of  S.  ellipsoideus  from  California 
grapes  and  of  cells  containing  spores  will  be  found  in  figures  5  and  6. 


8  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

The  main  part  of  all  wine  fermentations  is  carried  on  by  this  yeast. 
There  are  a  great  many  different  varieties  in  this  group  and  these  vary 
considerably  in  their  suitability  for  wine-making  purposes.  Modern 
methods  of  wine  making  aim  to  make  use  of  the  most  desirable 
varieties. 

2.  Saccharomyces  cerevisiae  (Beer  Yeast). — It  is  possible  that 
these  yeasts  occur  on  grapes  to  a  more  or  less  limited  extent,  depend- 
ing upon  the  proximity  to  vineyards  of  breweries  or  distilleries  using 
these  yeasts.  No  reference  has  been  found  in  the  literature  describing 
their  occurrence  on  grapes  and  none  have  been  found  on  California 
grapes.  There  is  probably  little  likelihood  of  their  being  present  on 
grapes  in  sufficient  numbers  to  influence  in  any  way  the  fermenta- 
tions. This  yeast  is  worthy  of  mention  in  connection  with  wine  mak- 
ing, however,  because  it  has  been  used  in  the  past  to  start  wine  fer- 
mentations. "Where  brewery  yeast  is  so  used,  it  produces  a  wine  of 
beerlike  flavor  and  low  alcohol  content  and  with  an  excess  of  unfer- 
mented  sugar.  Such  a  wine  is  not  palatable  and  is  very  liable  to  be 
attacked  by  bacteria  and  to  be  lost  through  bacterial  decomposition. 

3.  Saccharomyces  malei. — This  yeast  occurs  on  apples  and  is 
similar  to  8.  ellipsoideus,  but  usually  forms  less  alcohol.  It  was  not 
found  on  any  samples  of  grapes  examined,  but  probably  occurs  occa- 
sionally on  grapes. 

Wild  Yeasts. —  (1)  Saccharomyces  pastorianus. — This  group  of 
yeasts  is  characterized  by  its  elongate  or  sausage-shaped  appearance 
and  its  ability  to  form  spores.  Members  of  this  group  of  yeasts  have 
been  found  on  two  samples  of  California  grapes.  They  form  small 
amounts  of  alcohol  in  grape  must  and  at  the  same  time  usually  pro- 
duce undesirable  flavors  and  odors — usually  a  bitter  flavor.  The  mic- 
roscopical appearance  of  a  culture  of  S.  pastorianus  yeast  from  Cali- 
fornia grapes  is  shown  in  figures  3  and  6. 

2.  Saccharomyces  anomalus  (Willia  Yeasts). — The  yeasts  of  this 
group  are  characterized  by  the  production  of  hat-shaped  spores.  The 
appearance  of  such  spores  is  shown  in  figure  7.  The  members  of  this 
group  grow  rapidly  in  must  and  form  a  wrinkled  film.  They  carry 
on  a  weak  fermentation  with  the  production  of  small  amounts  of 
alcohol.  Liquids  fermented  by  these  yeasts  are  high  in  aromatic  com- 
pounds of  various  sorts  and  for  this  reason  their  use  has  been  sug- 
gested as  ;i  means  of  flavoring  various  fermented  beverages.  Cells 
from  a  ei i H  me  isolated  from  California  grapes  are  shown  magnified 
1000  diameters  in  figures  3  and  6. 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  9 

3.  Saccharomyces  ludwigii. — This  yeast  has  been  found  on  grapes 
grown  in  Europe.  In  microscopical  appearance  it  resembles  the 
S.  apiculatus  yeast,  but  is  much  larger.  It  was  not  found  on  Cali- 
fornia grapes. 

4.  Saccharomyces  marxianus. — This  yeast  has  been  reported  as 
being  present  on  grapes.  It  is  recognized  by  its  kidney-shaped  spores. 
It  was  not  found  on  the  samples  of  California  grapes  examined. 


Pseudo-Yeasts 

Apiculatus  Yeast. — The  apiculatus  yeast  (Saccharomyces  apicu- 
latus, Hansenia  apiculata)  is  recognized  by  the  peculiarly  pointed  ap- 
pearance of  many  of  its  cells  (fig.  3) .  According  to  most  authorities  it 
does  not  form  spores  and  so  is  placed  in  the  group  of  pseudo-yeasts. 
According  to  Lindner,  however,  it  forms  spores  in  drop  cultures,  one 
spore  per  cell.  It  carries  on  a  feeble  bottom  fermentation  in  grape 
must  but  does  not  have  the  power  to  ferment  saccharose,  maltose, 
or  lactose.  In  must  it  gives  from  0%  to  6%  alcohol  and  at  the  same 
time  produces  fruity  flavors  and  odors.  It  settles  more  slowly  and 
less  completely  than  8.  ellipsoideus.  Apiculatus  yeast  probably  does 
more  harm  in  wine  making  than  all  other  varieties  of  wild  yeasts  com- 
bined, because  of  its  large  numbers  on  grapes  and  its  very  rapid 
development  after  the  grapes  are  crushed.  It  develops  so  rapidly  that 
the  first  stages  of  most  natural  wine  fermentations  are  carried  on  by 
this  yeast  and  the  preliminary  fermentation  of  grapes  is  often  spoken 
of  as  the  "apiculate"  stage.  During  this  preliminary  fermentation 
it  produces  undesirable  flavors  and  aromas,  destroys  yeast  food  that 
should  have  gone  to  the  true  wine  yeast,  forms  compounds  deleterious 
to  the  vigorous  development  of  the  true  wine  yeast  and  gives  a  great 
many  cells  of  low  specific  gravity  that  settle  out  slowly  after  the  main 
fermentation  is  over.  After  the  S.  ellipsoideus  yeast  has  formed  8  to 
10  per  cent  alcohol  the  Apiculatus  yeast  is  killed  and  will  not  be  found 
in  the  living  state  in  the  finished  wine. 

Mycoderma  Types. — The  Mycoderma  forms  are  known  to  all  wine 
makers  as  "wine  flowers"  (fig.  4).  They  are  present  in  considerable 
numbers  on  uncrushed  grapes  and  have  been  found  on  nearly  all 
samples  of  California  grapes  so  far  examined.  In  pure  cultures  in 
liquids,  they  appear  as  white  films,  usually  more  or  less  deeply 
wrinkled.  From  time  to  time  portions  of  the  pellicle  are  detached 
and  fall  to  the  bottom  giving  in  time  a  large  amount  of  sediment.    A 


10  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

feeble  fermentation  is  carried  on  at  the  surface  of  the  liquid,  pro- 
vided that  it  contains  fermentable  sugar.  Under  the  microscope  the 
yeast  appears  as  cells  of  irregular  shape  and  size,  the  usual  form,  how- 
ever, being  elongate  or  sausage-shaped.  The  cells  tend  to  adhere 
together  in  groups  and  chains.  It  is  thought  that  the  cells  are  main- 
tained at  the  surface  of  the  liquid  by  means  of  small  bubbles  of  air 
occluded  between  them. 

In  pure  cultures  in  grape  must,  the  Mycoderma  yeasts  are  capable 
of  destroying  varying  amounts  of  sugar  with  the  production  of  small 
amounts  of  alcohol,  in  most  cases,  and  obnoxious  flavors  and  aromas. 
It  is  probable  that  in  many  natural  wine  fermentations  they  are  more 
or  less  active  at  the  beginning  of  the  fermentation.  They  undoubtedly 
cause  trouble  in  tanks  of  wine  that  are  not  kept  well  filled  during 
storage.     They  are  aerobic. 

Torula  Yeasts. — Under  the  heading  of  Torula  are  placed  most  of 
the  yeasts  that  do  not  fit  into  other  groups.  Consequently,  this  name 
covers  yeasts  of  wide  variation  in  properties.  As  a  group  they  do  not 
form  spores;  they  form  small  amounts  of  alcohol,  do  not  normally 
develop  as  a  film  growth,  and  the  form  considered  as  typical  is  spher- 
ical, although  there  is  considerable  variation  from  the  typical  form. 
They  are  found  on  grapes  fairly  commonly,  though  less  abundantly 
than  8.  apiculatus  and  Mycoderma.  Two  forms  from  California 
grapes  are  shown  in  figure  3. 

II.  PROPERTIES  OF  MOLDS  AND  BACTERIA  FROM 
CALIFORNIA  GRAPES 

Sources  of  Cultures 

In  1911  micro-organisms  were  isolated  from  grapes  received  at  the 
winery  of  J.  E.  Colton,  Martinez,  California,  and  from  grapes  picked 
at  the  University  Farm,  Davis.  Samples  were  obtained  in  1912  from 
Fresno  and  El  Centro.  The  properties  of  these  micro-organisms  were 
studied  more  or  less  in  detail.  Most  attention  was  paid  to  the  or- 
ganisms and  characteristics  that  were  of  importance  in  wine  making. 

Method  of  Separation. — The  materials  used  in  the  separation  of 
the  various  types  of  organisms  present  on  the  grapes  were  sterile 
grape  must  agar,  sterile  grape  must,  petri  dishes,  and  a  small  platinum 
inoculating  rod.  The  grape  must  agar  was  made  by  dissolving  20 
grams  of  agar  agar  in  1000  c.c.  of  boiling  water  to  which  was  added 
60  c.c.  of  grape  must  after  the  agar  had  dissolved.     It  is  necessary 


1918]  Cruess:    Fermentation  Organisms  of  California  Grapes 


11 


Figure    3 
Yeasts  from  California  grapes  magnified  1000  diameters: 

35.  S.  apiculatus  from  Davis  grapes. 
72.  S.  apiculatus  from  Acampo  grapes. 
37.  Torula  from  Davis  grapes. 

77.  Torula  from  Contra    Costa    County   grapes. 

36.  S.  pastorianus  from   Davis  grapes. 
69.  Willia   species  -from   Acampo   grapes. 


12  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

to  use  this  high  diluation  of  must  in  the  agar  in  order  that  the  agar 
will  not  be  hydrolyzed  by  the  acid  of  the  must.  At  this  dilution  the 
agar  contains  about  .05  per  cent  acid  as  tartaric.  This  should  permit 
the  growth  of  all  forms.  The  hot  agar  solution  was  filtered  through 
cotton  wool  and  filled  in  10  c.c.  portions  into  test  tubes  plugged  with 
cotton  wool.  It  was  sterilized  at  15  pounds  pressure  in  an  autoclave. 
The  grape  must  was  clarified  by  boiling  with  the  white  of  egg  and 
filtration.  The  clear  must  was  filled  into  plugged  test  tubes  and 
sterilized  at  100°  C.  The  Petri  dishes  were  sterilized  in  packages  of 
three  by  dry  heat.    The  type  of  dish  used  is  shown  in  figure  3. 

Samples  of  the  grapes  received  at  the  winery  were  taken  from  the 
center  of  the  boxes  and  crushed  into  sterile  containers.  A  small 
amount  of  the  must  was  in  each  case  transferred  by  a  sterile  platinum 
wire  to  a  tube  of  melted  agar  kept  at  40°  C  to  45°  C,  and  further 
dilutions  were  made  by  transfer  to  other  tubes  of  agar. 

The  organisms  were  separated  by  growth  on  agar  must.  They  were 
purified  by  replating  on  the  same  medium.  Subcultures  were  then 
made  in  sterile  must,  on  agar  slants  and  permanent  stock  cultures  were 
made  in  sterile  10  per  cent  cane  sugar  in  Freudenreich  flasks. 

The  pure  cultures  obtained  in  this  way  are  discussed  in  groups, 
the  members  of  each  group  having  certain  characteristics  in  common. 
No  attempt  has  been  made  so  far  to  study  the  molds  and  bacteria  in 
detail  and  for  this  reason  they  have  simply  been  listed  with  a  few 
words  of  explanation. 

Molds 

Penicillium  Species  (probably  Penicillium  expansum) . — Found  on 
nearly  all  samples  examined ;  produces  fructifications  bearing  numer- 
ous round  conidia  as  shown  in  figure  1 ;  forms  sclerotia,  resistant 
vegetative  forms,  in  old  cultures;  no  fermentation  in  must,  but  grows 
vigorously  in  all  culture  media  tested;  giving  a  characteristic  moldy 
odor. 

Penicillium  Species  of  Olive  Green  Color. — Forms  ellipsoidal  coni- 
dia ;  very  common  on  California  grapes  and  also  found  in  samples  of 
pasteurized  unfermented  grape  juice  that  had  molded  after  pasteuri- 
zation. 

Aspergillus. — Two  forms:  (a)  produces  small,  smooth,  black  coni- 
<li;i  ;  (b)  produces  large  black  conidia  with  spikelike  projections.  The 
Jippearance  of  the  one  yielding  the  smooth  conidia  is  shown  in  figure  1. 

Botrytis  (probably  H.  cinerea). — Found  on  many  samples  of  grapes 


1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


13 


Figure  4 
Yeasts  from  California  grapes: 

65.  Mycoderma  yeast  from  Acampo  grapes. 
68.  Mycoderma  yeast  from  Acampo  grapes. 

70.  Mycoderma  yeast  from  Acampo  grapes. 

71.  Mycoderma  yeast  from  Contra  Costa  Grapes. 
73.  Mycoderma  yeast  from  Acampo  grapes. 

76.  Mycoderma  yeast  from  Acampo  grapes. 


14  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

after  the  early  rains  of  1912.  It  gives  the  characteristic  Botrytis 
growth  on  the  grapes  but  is  very  reluctant  to  grow  on  any  of  the 
ordinary  culture  media,  the  best  artificial  medium  found  so  far  being 
grape  must  gelatin  made  up  by  dissolving  gelatin  in  undiluted  grape 
must.  The  appearance  of  this  mold  from  grapes  grown  at  the  vine- 
yard of  J.  Swett  and  Son,  Martinez,  is  shown  in  figure  1. 

Mucor. — Color,  gray;  spores,  shortly  ellipsoidal.  Does  not  pro- 
duce fermentation  in  grape  must.  Its  microscopical  appearance  is 
given  in  figure  1. 

Dematium  Variety. — Grows  vigorously  in  must  giving  a  slimy  mass 
of  colorless  cells  and  a  surface  growth  of  black  cells  arranged  in  long 
chains.  Found  in  large  numbers  on  grapes  examined  in  1912.  Does 
not  cause  fermentation  in  must.  Microscopical  appearance  is  given 
in  figure  2. 

Monilia  Species. — Colonies  on  agar  resemble  yeast  colonies  with 
rootlike  projections  into  the  depths  of  the  medium.  Gives  a  feeble 
fermentation  in  grape  must,  followed  by  a  mycelial  growth  on  the 
surface  of  the  liquid ;  in  old  cultures,  part  of  the  mycelium  becomes 
olive  green  (fig.  2). 

Dark  Green  Mold  (unidentified). — Found  growing  profusely  as  a 
parasitic  fungus  on  skins  of  grapes  after  the  early  rains  in  fall  of 
1912 ;  grows  equally  well  on  artificial  media  in  the  laboratory,  so  ma}7 
be  termed  a  faculatative  parasite,  It  causes  no  visible  fermentation 
in  must  and  is  odorless.  Its  microscopical  appearance  is  given  in 
figure  1.     It  may  possibly  be  a  Cladosporium  form. 

Bacteria 

Vinegar  Bacteria. — The  only  forms  of  bacteria  so  far  found  on 
grapes  examined  at  the  laboratory  or  winery  have  been  vinegar 
bacteria.  They  were  observed  on  two  different  lots  of  grapes,  both  of 
which  had  been  shipped  long  distances  and  were  in  a  badly  broken 
and  moldy  condition.  Both  cultures  grew  rapidly  in  grape  must 
giving  a  heavy  tough  film.  One  culture  gave  bacteria  of  medium 
length  that  tended  to  form  in  chains,  while  the  other  developed  in 
the  form  of  very  short  rods  usually  grouped  in  pairs  (fig.  2). 

"Tourne"  Bacteria. — The  form  shown  in  figure  2  is  found  in  wines 
quite  often,  but  has  not  been  met  with  on  California  grapes.  It  grows 
in  the  absence  of  air  and  gives  a  "mousey"  taste  to  badly  affected 
wines  (fig.  2).  This  organism  is  otherwise  known  as  Bacterium 
mannito-poeum.    It  occurs  in  vinegar  and  fermented  fruit  juices. 


1918]  Critess:   Fermentation  Organisms  of  California  Grapes 


15 


Figure    5 
Yeasts  from  California  grapes: 

78.  Mycoderma  yeast  from  Contra  Costa  County  grapes. 
64.  S.  ellipsoideus  from  Acampo    grapes. 

66.  S.  ellipsoideus  from  Davis   grapes. 

67.  S.  ellipsoideus  from  Contra   Costa   County   grapes. 

74.  S.  ellipsoideus  from  Acampo  grapes. 

75.  S.  ellipsoideus  from      Contra    Costa   County  grapes. 


16  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

III.    CHARACTERISTICS    OF   YEASTS    FROM    CALIFORNIA 

GRAPES 

Methods  of  Study 

The  yeasts  isolated  from  grapes  in  1911  according  to  the  methods 
given  on  preceding  pages  were  studied  in  accordance  with  the  follow- 
ing outline.  From  the  tests  made,  it  was  possible  to  determine  the 
specific  character  of  the  yeasts  and  to  obtain  a  fair  idea  as  to  their 
suitability  or  unsuitability  for  wine  making  purposes. 

Morphology. —  (1)  Macroscopical  Appearance. — In  must,  beer  wort, 
nutrient  dextrose,  saccharose,  and  lactose  solutions ;  on  agar  must  and 
on  gelatin  must. 

(2)  Microscopical  Appearance. — Size,  form,  and  general  appear- 
ance from  five  days'  growth  in  grape  must.  Microscopical  appearance 
of  agar  colonies. 

(3)  Spore  Formation. — Observed  by  placing  the  sediment  from 
vigorous  cultures  on  gypsum  blocks  partly  immersed  in  water  in  wide 
mouthed  bottles  plugged  with  cotton  wool.  This  arrangement  gives 
the  necessary  conditions  of  aeration,  poor  food  supply,  and  moisture. 
Tests  were  made  of  spore  formation  at  22°  C  and  28°  C. 

Fermentation  Tests. —  (1)  Rates  of  Fermentation. — The  rates  of 
fermentation  in  grape  musts  of  23.01%  and  29.75%  Balling,  in  beer 
wort,  nutrient3  dextrose  solution,  nutrient  saccharose  solution  and 
nutrient  lactose  solution  were  determined  on  100  c.c.  portions  of  the 
above  liquids  by  noting  the  loss  in  weight  during  fermentation  after 
inoculation  with  pure  cultures  of  the  organism  under  observation. 
The  tests  were  carried  out  at  33°  C.  The  loss  in  weight  is  due  to  the 
formation  of  carbon  dioxide  during  fermentation.  The  escape  of  this 
gas  causes  the  change  in  weight  by  which  the  fermentation  may  be 
followed.    The  main  chemical  reaction  involved  is  the  following : 

C  H    O   =  2CO    +  2C  H  OH 

6      12     6  2     '  2      5 

A  check  flask,  which  was  not  inoculated,  was  used  as  a  means  of 
obtaining  the  loss  in  weight  due  to  evaporation.  The  figures  given  in 
the  tables  are  corrected  for  this  loss.  Points  representing  the  time  in 
hours  reckoned  from  the  time  of  inoculation  and  also  representing  the 


— i 

8  The  nutrient  solutions  used  for  the  dextrose,  saccharose,  and  lactose  con- 
sisted of  .01  grams  magnesium  sulfate,  .5  grams  of  dipotassium  phosphate, 
10  grams  Witte  's  pepton  dissolved  in  1000  c.c.  of  water.  To  a  portion  of  this 
solution  was  added  15%  dextrose,  to  a  second  part  15%  saccharose,  and  to  the 
third,  15%  lactose. 


1918] 


Cruess:   Fermentation  Organisms  of  California  Grapes 


17 


66 

W/  TH  SPOftCS 


79 


WITH  SRORES- 


WITH 


69 

S/=OF?£S. 


Figure  6 
Yeasts  from   California  grapes   showing  spore   formation: 
79.  S.  ellipsoideus  from  Contra  Costa  County,  California. 
66.  S.  ellipsoideus  from  Davis  grapes  showing  spores. 
75.  S.  ellipsoideus  from  Contra  Costa  County  grapes,  showing  spores. 
79.  S.  ellipsoideus  from  Contra  Costa  County  grapes,  showing  spores. 
36.  S.  pastorianus  from  Davis  grapes,  showing  spores. 
69.  Willia  from  Acampo  grapes,  showing  spores. 


18  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

loss  in  weight  have  been  plotted  on  diagrams  and  the  individual  points 
connected  to  give  "fermentation  curves."  These  curves  show  at  a 
glance  the  character  of  the  fermentations  and  serve  as  convenient 
means  of  comparison. 

(1)  Products  of  Fermentation. — The  products  of  fermentation  in 
the  solutions  given  above  were  determined  after  the  rates  of  fermenta- 
tion were  ascertained.  Similar  tests  were  made  in  grape  must  of  30° 4 
Balling  fermented  by  the  various  yeasts  at  24°  C. 

Yeasts  Studied 

The  yeasts  have  been  taken  up  in  the  following  order :  (1)  S.  apicu- 
latus  yeasts,  (2)  Mycoderma  forms,  (3)  Torula  yeasts,  (4)  S.  pastor- 
ianus  and  Willia  yeasts,  and  (5)  S.  ellipsoideus  yeasts.  The  char- 
acteristics of  each  have  been  discussed  according  to  the  outline  given 
above. 

APICULATUS  YEASTS  FROM  CALIFORNIA  GRAPES 

Two  apiculatus  yeasts  were  studied.  One  of  these  was  isolated 
from  grapes  grown  at  Davis  and  the  other  from  grapes  grown  at 
Acampo.  The  morphology  and  biological  characteristics  were  studied 
as  previously  outlined. 

Microscopical  appearance  is  based  on  cells  from  five  days'  growth 
in  grape  must  of  15%  Balling.  The  agar  used  for  plate  cultures  con- 
sisted of  2  grams  of  agar  agar,  60  c.c.  of  must,  and  1000  c.c.  of  water. 
The  gelatin  medium  consisted  of  15  grams  of  gelatin  to  each  100  c.c. 
of  grape  must. 

Organism  35.    Apiculatus  from  Grapes  Grown  at  Davis 

Morphology. — Microscopical  Appearance. — Majority  of  cells  lemon- 
shape.  Cells  broader  and  larger  per  cent  apiculate-shape  than  in  case 
of  yeast  no.  72. 

Size. — Average,  4.5/*  x  2.4//,.  Maximum,  7.5/a  x4.5^.  Minimum, 
3/x  x  1.5/x. 

Spore  Formation. — No  spores  at  22°  C  or  28°  C. 

Colonics  on  Agar  Agar. — Flat.  Elevation  uniform.  Smaller  than 
8.  elUpsoideus  colonics.  Semitranslucent.  Edges  smooth  and  entire. 
Under  octfcroscope  the  edges  of  the  colonics  are  entire. 


4  Degrees  Balling   indicates  total   dissolved  solids  in  grams  per   100  grams, 
as  cane  Bugar. 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  19 

Colonies  on  Gelatin. — Small.  Gelatin  is  slowly  liquefied,  giving 
craterlike  depressions.    Edges  entire. 

Growth  in  Liquid  Media. — In  grape  must,  beer  wort,  nutrient 
dextrose,  and  saccharose  solutions,  rapid  but  fermentation  feeble. 
Growth  in  lactose  slight  and  no  fermentation.  Sediment  and  growth 
in  liquid,  fine  grained.  Cells  settle  slowly.  Liquid  becomes  fruity 
in  flavor  and  odor. 

Organism  72.    Apicnlatus  from  Grapes  Grown  at  Acampo 

Morphology. — Microscopical  Appearance. — Majority  of  cells  sau- 
sage-shaped or  spherical.  Only  a  few  typical  apiculate  cells.  De- 
cidedly different  from  no.  35. 

Size. — Average,  4.5/x  x  2.7/*.  Maximum,  6/x  x  2.7/x.  Minimum 
2.2/XX1.5/*. 

Spore  Formation. — No  spores  at  28°  C  or  22°  C. 

Colonies  on  Agar  Agar. — Small.  Edges  undulate  or  indented. 
Centers  slightly  elevated.  Opaque  and  porcelainous.  Under  low 
power,  edges  entire.    Different  from  no.  35. 

Colonies  on  Gelatin. — Small.  Edges  branchlike.  Waxy  luster. 
Edges  under  microscope  lacerate,  i.e.,  saw-tooth  appearance.  Gelatin 
slowly  liquefied  to  form  craterlike  depressions  around  colonies ;  whole 
plate  liquefied  in  few  days. 

Growth  in  Liquid  Media. — In  grape  must,  beer  wort,  and  nutrient 
dextrose,  growth  is  rapid.  Almost  no  growth  in  saccharose  and  lactose 
nutrient  solutions.  Deposit  and  growth  in  liquid  fine  grained.  Settles 
very  slowly.     Fruity  odor  and  flavor  in  fermented  liquid. 

Rates  of  Fermentation,  Various  Media. 
The  following  solutions  were  used : 
{a)   Grape  must,  23.01°  Balling. 

(b)  Grape  must,  29.75°  Balling. 

(c)  Sweet  beer  wort,  14°  Balling. 
(d)5  Nutrient  dextrose,  15%  dextrose, 
(e)   Nutrient  saccharose,  15%  saccharose. 
(/)    Nutrient  lactose,  15%  saccharose. 


5  The  nutrient  solutions  (d),  (e) ,  (/),  contained  .01  grams  MgS(X  .5  grams 
K2HP04,  10  grams  Witte's  pepton,  per  1000  c.c.  water,  and  150  grams  of  the 
respective  sugars  per  1000  c.c. 


20  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

One  hundred  cubic  centimeters  of  the  above  solutions  were  steril- 
ized in  small  plugged  flasks.  The  solutions  were  inoculated  in  each 
instance  with  one  platinum  loopful  of  a  vigorous  culture.  Non- 
inoculated  checks  were  used  to  correct  for  loss  due  to  evaporation. 
The  inoculated  flasks  were  placed  in  an  incubator  at  33°  C. 

The  flasks  were  weighed  at  intervals  during  fermentation.  The 
loss  in  weight  corrected  for  loss  due  to  evaporation  was  taken  as  a 
measure  of  the  rate  of  fermentation. 

TABLE  1 

Data  on  Eates  of  Fermentation  of  Yeasts  Numbers  35  and  72  (S.  apiculatus) 
in  Various  Sugar  Solutions 

Must  of 


Yeast 
lumber 

35 

Time  in 
hours 

46 

23°  Balling,  Concentrated 
loss  in           must,  loss 
grams            in  grams 

1.85                   .33 

Wort  loss, 
in  grams 

.35 

Dextrose 
loss,  in 
grams 

.45 

Saccharose 
loss,  in 
grams 

.03 

Lactose 
loss,  in 
grams 

.00 

35 

73 

2.84              1.10 

.45 

.65 

.02 

.00 

35 

96 

3.26             2.05 

.47 

.79 

.01 

.00 

35 

118 

3.42              2.53 

.47 

.93 

.01 

.00 

35 

167 

3.54              2.90 

.49 

1.13 

.00 

.00 

35 

215 

3.54              2.95 

.57 

1.35 

.03 

.00 

35 

239 

3.57             2.98 

.58 

1.40 

.00 

.00 

72 

27 

.18               .01 

.35 

.10 

.00 

.00 

72 

52 

1.09                .19 

.56 

.33 

.00 

.00 

72 

77 

1.92                .42 

.59 

.83 

.00 

.00 

72 

125 

2.75              1.15 

.69 

.98 

.00 

.00 

72 

167 

2.84              1.32 

.96 

.00 

.00 

72 

244 

3.18              2.05 

.97 

1.21 

.00 

.00 

72 

341 

3.50              2.47 

1.10 

1.42 

.00 

.00 

72 

421 

3.55             2.53 

1.13 

1.44 

.00 

.00 

From  an  examination  of  the  data  given  in  table  1  and  of  the 
fermentation  curves  in  the  accompanying  diagram,  it  is  evident  that 
both  yeasts  have  very  low  fermenting  power  and  slow  rates  of  fer- 
mentation. In  grape  must  at  33°  C,  yeast  no.  35  produces  a  more 
rapid  fermentation  than  yeast  no.  72,  but  in  beer  wort  and  dextrose 
solutions  the  two  yeasts  are  similar.  The  concentrated  must  of  29.75° 
Balling  has  a  greater  retarding  effect  on  yeast  no.  72  than  on  yeast 
no.  35.  Neither  yeast  fermented  saccharose  or  lactose.  Because  of 
their  difference  of  morphology  and  in  the  character  of  their  fermenta- 
1  ions,  it  is  justifiable  to  designate  them  as  two  different  varieties  of 
apiculatus  yeast. 

Attenuation  and  Alcohol  Formation. — The  liquids  listed  in  table  2 
were  analyzed  after  fermentation.  The  term  "attenuation"  in  the 
accompanying  table  refers  to  the  loss  in  sugar  during  fermentation. 
The  alcohol,  theoretical  and  observed,  is  reported  as  volume  per  cent. 


1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


21 


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TABLE  2 

Yeasts  Numbers  35  and  72   (S.  apiculatus).     Attenuation  and  Yields 
Alcohol  in  Must,  Beer  Wort,  Dextrose,  Saccharose,  and 
Lactose  Solutions 


Yeast 
Xo. 

72 

72 

72 

72 

72 

72 


Attenuation 
in  grams 
per  100 
c.c. 


Medium 

Must  of  23°  Balling  3.81 

Must  of  29?75  Balling  1.20 

Beer   wort    of    14?56   Balling  1.61 

Dextrose  solution,  14?9  Ball.  2.73 

Saccharose    sol.,    13?73    Ball.  .78 

Lactose  solution,  15?16  Ball.  .31 


Theoretical 
yield  of 
alcohol, 
per  cent 

2.50 

.79 
1.11 
1.79 

.53 

.20 


Observed 
yield  of 
alcohol, 
per  cent 

1.00 

.30 

.30 

.65 

.00 

.00 


35     Must   of   23°    Balling   8.50 

35     Must  of  29?75  Balling  6.15 

35  Beer   wort   of    14?56   Balling  1.35 

35  Dextrose  solution,  14?9  Ball.  3.25 

35  Saccharose    sol.,    13? 73    Ball.  .00 

35  Lactose  solution,  15?16  Ball.  .00 


5.58 

4.03 

.93 

2.13 

.00 

.00 


3.05 
2.35 

.65 
.00 
.00 


Per  cent  of 
theoretical 

yield  ob- 
yield 

obtained 

40.0 

37.9 

27.6 

36.3 
.00 
.00 


54.5 
58.3 


30.5 


22  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

The  results  show  that  in  most  cases,  less  than  50%  of  the  alcohol, 
theoretically  obtainable  from  the  sugar  fermented,  was  actually  formed 
by  the  two  yeasts.  For  example,  yeast  no.  35  destroyed  3.81%  sugar 
and  should  have  produced  2.5%  alcohol  in  must  of  23°  Balling,  while 
the  actual  yield  was  only  1%  or  40%  of  the  amount  that  was  theo- 
retically possible.  True  wine  yeasts  of  the  8.  ellipsoideus  type  pro- 
duce at  least  90%  of  the  theoretical  yield  in  grape  must. 

Since  the  yields  of  alcohol  are  so  low  and  the  flavor  of  the  fer- 
mented liquids  is  not  desirable,  the  two  yeasts  may  be  considered 
of  little  use  for  industrial  purposes.  Their  importance  lies  in  their 
power  to  cause  trouble  in  wine  making  by  growing  in  natural  fer- 
mentations before  the  true  wine  yeast  has  developed.  In  this  way  they 
may  injure  the  flavor  and  clearing  quality  of  the  wine  and  favor 
"stuck"  tanks  by  the  production  of  compounds  that  are  injurious 
to  the  8.  ellipsoideus  yeast.  Because  of  their  very  common  occurrence 
in  large  numbers  on  California  grapes,  measures  should  always  be 
taken  to  discourage  their  growth. 

MYCODERMA  FORMS  FROM  CALIFORNIA  GRAPES 

Under  Mycoderma  forms,  seven  film  forming  organisms  have  been 
described  in  the  following  pages.  Some  of  these  organisms  are  yeast- 
like in  appearance  and  in  their  fermentation  properties ;  other  re- 
semble molds  more  closely  than  they  do  yeasts.  All  have  the  common 
properties  of  forming  heavy  films  on  nutrient  liquids  and  of  not 
forming  spores. 

Organism  65.    Mycoderma  from  Acampo  Grapes. 

Morphology. — Microscopical  Appearance. — Sausage-shape  to  fila- 
mentous.   Tends  to  grow  in  mycelial  threads  (fig.  4). 

Size.  —  Average,  9^  x  2.5/x.  Maximum,  40/x  x  2.5/x.  Minimum, 
2/x  x  1.5/x. 

Spore  Formation. — No  spores  at  22°  C  and  28°  C. 

Colonies  on  Agar  Agar. — Small  and  growth  slow.  Powdery  ap- 
pearance. Color,  white.  Flat.  Growth  at  surface  only.  Edges  made 
up  of  mycelial  threads.  Under  low  power,  colonies  are  made  up  of 
chains  of  long  cells.     These  spread  over  the  surface  of  the  agar. 

Colonies  on  Gelatin. — More  vigorous  than  on  agar.  Concentric 
ring  growth.  Powdery  white.  Flat.  Under  microscope,  same  as  on 
agar.    Gelatin  slowly  liquefied. 


1918]  Cruess:    Fermentation  Organisms  of  California  Grapes  23 

Growth  on  Liquid  Media. — Grows  profusely  on  surface  of  grape 
must,  beer  wort,  dextrose,  saccharose  and  lactose  solutions,  but  does 
not  produce  visible  fermentation  in  any  of  these  liquids.  Film  remains 
permanently  on  the  surface.  Does  not  sink.  Film  is  very  tough  in 
texture. 

Organism  68.    Mycoderma  from  Grapes  Grown  at  Acampo,  California 

Morphology. — Microscopical  Appearance.  —  Predominating  form 
sausage-shaped.  Cells  vary  from  spherical  to  long-rod  shaped  cells 
(fig.  4). 

Size. — Average,  7.5/x  x  3.75/x.  Maximum,  15/*  x  3.75/x.  Minimum, 
3/x  x  S/x. 

Spore  Formation. — No  spores  formed. at  22°  C  or  28°  C. 

Colonies  on  Agar  Agar. — Large.  Edges  entire.  Waxy  luster. 
Centers  of  colonies  slightly  elevated.  Edges  entire.  Opaque.  Under 
low  power  of  microscope,  internal  structure  granular  and  edges  are 
surrounded  by  long  chains  of  cells  of  ellipsoidal  shape. 

Colonies  on  Gelatin.  Filmy,  translucent  colonies,  that  rapidly 
cover  the  whole  surface.  Under  microscope  the  colonies  are  made  up 
of  chains  of  cells. 

Growth  in  Liquid  Media. — Heavy  growth  in  grape  must,  beer  wort, 
and  dextrose  with  formation  of  large  sediment,  the  volume  of  which 
may  be  equal  to  10  per  cent  of  the  total  volume  of  the  liquid.  Growth 
slow  in  lactose  and  saccharose  solutions.  Visible  fermentation  very 
slow  in  grape  must,  beer  wort  and  dextrose  solution  at  first,  but  finally 
becomes  vigorous  in  must  and  dextrose  solutions.  No  visible  fermenta- 
tion in  lactose  and  saccharose. 

Organism  70.     Mycoderma  from  Acampo  Grapes 

Morphology. — Microscopical  Appearance. — Usual  form,  sausage- 
shape,  but  vary  from  spherical  to  sausage-shaped.  Cells  are  usually 
grouped  in  branching  chains  (fig.  4). 

Size. — Average,  7/*  x  2/x.  Maximum,  18/x.  x  2/x.  Minimum,  1.5/x  x  1.5^. 

Spore  Formation. — No  spores  formed  at  22°  C  and  28°  C. 

Colonies  on  Agar  Agar.  Large.  Center  elevated.  Surface  cor- 
rugated by  ridges  radiating  from  centers  of  colonies.  Centers  of 
colonies  brown ;  edges  yellowish  white.  Edges  indented.  Low  power 
of  microscope  shows  chains  of  cells  growing  from  edges  of  colonies; 
this  growth  is  beneath  the  surface  of  the  agar  (see  plate  2). 


24  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

Colonies  on  Gelatin. — Large.  Centers  convex.  Colonies  flatten 
near  edges  and  become  filmy  in  appearance.  Edges  entire  under 
microscope.     Gelatin  slowly  liquefied. 

Growth  in  Liquid  Media. — Heavy,  wrinkled  surface  growth  wrhich 
frequently  falls  to  bottom  forming  voluminous  sediment.  Fermenta- 
tion slow.     No  fermentation  in  lactose. 

Organism  71.    Mycoderma  from  Contra  Costa  County  Grapes 

Morphology. — Microscopical  Appearance.  —  Predominating  form 
sausage-shaped.    Varies  from  spherical  to  sausage-shape  (fig.  4). 

Size. — Average,     6.9/x  x  2/x.       Maximum,     18/x  x  2/x.       Minimum, 

1.5/X  X  1.5/X. 

Spore  Formation.— No  spores  at  22°  C  or  28°  C. 

Colonies  on  Agar  Agar. — Large.  Flat.  Spreading.  Edges  lobate. 
Edges  entire  under  microscope  (see  plate  2). 

Colonies  on  Gelatin. — Flat.  Filmy.  Translucent.  Rapidly  cover 
entire  surface  of  the  gelatin.    Slow  liquefaction  (see  plate  2). 

Growth  in  Liquid  Media. — A  wrinkled,  loosely  coherent  film  on  all 
liquids  tested.  Films  frequently  sink  and  are  quickly  replaced  by  new 
growth.  Fermentation  in  must,  beer  wort,  and  dextrose  fairly  vigor- 
ous, but  extends  over  long  period.  No  perceptible  fermentation  in  sac- 
charose and  lactose. 

Organism  73.    Mycoderma  from  Grapes  Grown  at  Acampo 

Morphology. — Microscopical  Appearance. — Usual  form  sausage- 
shaped.    Varies  from  spherical  to  sausage-shaped  (fig.  4). 

Size. — Average,  6.7/*.  x  2.5/x.  Maximum,  18/x  x  3/x.  Minimum, 
3/x  x  1.5/*. 

Spore  Formation. — No  spore  formation  at  22°  C  and  28°  C. 

Colonies  on  Agar  Agar. — Centers  elevated.  Edges  flat  and  ser- 
rated. Shape  irregular.  Under  the  microscope  chains  of  cells  are 
seen  to  radiate  from  the  edges  of  the  colonies.  The  chains  are  made 
up  of  long  cells  with  pairs  of  short  ellipsoidal  cells  at  the  junctures 
of  the  long  cells. 

Colonies  on  Gelatin. — Flat.  Smooth.  Filmy.  Translucent.  Gela- 
liti  rapidly  covered  by  a  filamentous  growth.  Gelatin  is  slowly 
softened  to  a  syrupy  consistency. 

Growth  in  Liquid  Media. — Growth  vigorous  in  all  liquid  media 
tested.     Vigorous  fermentation  in  grape  must  and  dextrose  solution. 


1918]  Cruess:    Fermentation  Organisms  of  California  Grapes  25 

During  fermentation  a  heavy  wrinkled  film  is  formed  with  a  moderate 
amount  of  sediment  growth.  After  fermentation  most  of  the  film 
growth  settles  to  the  bottom.  Fermentation  is  more  vigorous  and  film 
formation  is  not  so  well  developed  as  in  no.  70. 

Organism  76.    Mycoderma  from  Grapes  Grown  at  Acampo 

Morphology. — Microscopical  Appearance. — Sausage-shaped  to  fila- 
mentous (fig.  4). 

Size. — Average,  10.5/x  x  2.1/x.  Maximum,  75/*.  x  2.5/*.  Minimum, 
3/x  x  2>fx. 

Spore  Formation. — No  spores  formed  at  22°  C  or  28°  C. 

Colonies  on  Agar  Agar. — Large.  Flat.  Surface  smooth.  Centers 
slightly  elevated.    Edges  smooth  and  entire  under  microscope. 

Colonies  on  Gelatin. — Large.  Resinous.  Translucent.  Surface  of 
colonies  deeply  corrugated.  Mycelial  projections  from  edges  visible 
to  naked  eye.  Surface  glistening  and  slimy.  Under  microscope  edges 
are  fringed  by  branching  chains  of  very  long  cells. 

Growth  in  Liquid  Media. — Growth  in  all  media  tested  consists  first 
of  powdery  white  colonies.  These  coalesce  to  form  a  resinous  heavy 
film  resembling  a  growth  of  vinegar  bacteria.  The  growth  sinks  after 
several  weeks  and  is  replaced  by  a  second  film.  No  visible  fermenta- 
tion in  any  liquids  tested. 

Organism  78.    Mycoderma  from  Contra  Costa  County  Grapes 

Morphology. — Microscopical  Appearance.  —  Sausage-shaped  cells 
predominate.    Very  long  filamentous  cells  not  uncommon  (fig.  4) . 

Sige. — Average,  10.5/x  x  3/x.  Maximum,  37.5/a  x  3/x.  Minimum, 
4.5/x  x  2{x. 

Spore  Formation.— No  spores  at  22°  C  and  28°  C. 

Colonies  on  Agar  Agar. — Large  and  flat.  Centers  slightly  elevated. 
Waxy  luster.  Edges  show  hazy  growth  of  mycelium.  Under  micro- 
scope colonies  are  seen  to  be  surrounded  by  radiating  mycelial  threads. 

Colonies  on  Gelatin — Large.  Flat.  Smooth.  Translucent.  Rapidly 
cover  the  entire  surface  of  the  gelatin.  Gelatin  rapidly  softened  to  a 
syrupy  consistency  (see  plate  2). 

Growth  in  Liquid  Media. — In  grape  must,  beer  wort,  and  dextrose, 
solutions,  growth  is  rapid  and  fermentation  vigorous;  in  saccharose 
growth  vigorous  and  fermentation  slow ;  little  growth  in  lactose.  Con- 
sists chiefly  of  surface  growth.     Small  sediment. 


26  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Fermentation  Tests  of  Mycoderma  Forms  from  California  Grapes. 

The  data  obtained  by  noting  the  loss  in  weight  at  33°  C  of  flasks 
containing  100  c.c.  of  grape  must  23.01°  Balling,  grape  must  29.8° 
Balling,  beer  wort  14°  Balling,  dextrose  15°  Balling,  saccharose  15° 
Balling,  and  lactose  15°  Balling  solutions,  respectively,  after  inocula- 
tion with  pure  cultures  of  the  above  yeasts  appear  in  table  3.  The 
attenuation,  representing  the  sugar  destroyed,  together  with  the  yields 
of  alcohol  in  the  various  sugar  solutions  tested  are  given  in  table  3. 
After  each  table  the  results  of  the  tests  are  discussed.  The  nutrient 
solutions  used  were  the  same  as  those  described  for  yeasts  35  and  72. 
See  description  of  apiculatus  yeasts. 

TABLE  3 
Fermentation  Tests  of  Mycoderma  Forms  from  California  Grapes 

Dextrose      Saccharose        Lactose 


Yeast 
number 

Time  in 
hours 

Must  of 

23°  Balling, 

loss  in 

grams 

Must  of 

29°  Balling, 

loss,  in 

grams 

Wort  loss, 
in  grams 

14  per  cent 
solution 

loss,  in 

grams 

15  per  cent 

solution 

loss,  in 

grams 

15  per  cent 

solution 

loss,  in 

grams 

15  per  cent 

solution 

68 

18 

.00 

.00 

.04 

.07 

.00 

.00 

68 

42 

.70 

.01 

.21 

.31 

.10 

.00 

68 

66 

1.51 

.24 

.38 

.93 

.10 

.00 

68 

92 

2.45 

.62 

.37 

2.10 

.10 

.00 

68 

123 

3.35 

1.32 

.38 

2.90 

.10 

.00 

68 

165 

5.80 

2.00 

.40 

5.89 

.10 

.03 

68 

236 

8.45 

3.30 

.42 

6.40 

.10 

.03 

68 

284 

8.48 

3.80 

.50 

6.40 

.10 

.03 

68 

314 

8.48 

3.80 

.51 

.10 

.03 

65 

18 

.00 

.00 

.00 

.00 

.00 

.00 

65 

42 

.03 

.00 

.00 

.00 

.01 

.00 

65 

66 

.03 

.00 

.03 

.00 

.01 

.00 

65 

92 

.03 

.00 

.08 

.04 

.01 

.00 

65 

123 

.03 

.04 

.08 

.09 

.07 

.03 

65 

165 

.03 

.04 

.12 

.09 

.07 

.03 

65 

236 

.03 

.04 

.17 

.09 

.07 

.03 

70 

18 

.02 

.03 

.00 

.10 

.00 

.00 

70 

42 

.10 

.05 

.06 

.18 

.20 

.00 

70 

66 

.38 

.08 

.33 

.41 

.20 

.06 

70 

92 

.80 

.38 

.42 

.25 

.05 

70 

123 

1.90 

.08 

.80 

1.30 

.60 

.20 

70 

165 

2.47 

.83 

1.00 

1.65 

.90 

.20 

70 

236 

2.57 

.95 

1.00 

1.65 

.90 

.30 

71 

46 

.85 

.35 

.25 

.00 

.00 

71 

73 

1.85 

1.17 

.34 

.46 

.00 

71 

96 

2.70 

2.19 

.41 

.00 

71 

118 

3.74 

.■',.19 

.42 

2.31 

.01 

71 

167 

5.47 

3.89 

.42 

4.20 

.00 

.00 

71 

215 

6.40 

5.90 

.44 

.04 

71 

239 

6.85 

6.40 

.45 

.04 

1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


27 


TABLE   3- 

-(Continued) 

Yeast 
lumber 

Time  in 
hours 

Must  of 
23°  Ballin 
loss  in 
grams 

Must  of 
g,   29°  Balling 
loss,  in 
grams 

Wort  loss, 
in  grams 

14  per  cent 
solution 

Dextrose 

loss,  in 

grams 

15  per  cent 

solution 

Saccharose 
loss,  in 
grams 

15  per  cent 
solution 

Lactose 

loss,  in 

grams 

15  per  cent 

solution 

73 

27 

.20 

.06 

.18 

.66 

.10 

.00 

73 

52 

1.09 

.61 

.34 

1.79 

.28 

.00 

73 

77 

2.26 

1.56 

.49 

3.21 

.58 

.00 

73 

125 

4.14 

3.34 

.59 

4.11 

.93 

.00 

73 

167 

5.69 

4.59 

.61 

5.72 

1.19 

.00 

73 

244 

9.25 

6.62 

.72 

7.07 

1.41 

.00 

73 

341 

9.32 

8.24 

.87 

7.35 

1.52 

.00 

73 

421 

9.52 

9.65 

.92 

.00 

76 

27 

.00 

.04 

.00 

.00 

.00 

.00 

76 

52 

.04 

.08 

.04 

.07 

.00 

76 

77 

.00 

76 

125 

.00 

76 

167 

.31 

.13 

.24 

.20 

.00 

76 

244 

.42 

.39 

.46 

.41 

.00 

76 

341 

.62 

.69 

.62 

.50 

.00 

76 

421 

.64 

.69 

.70 

.54 

.00 

78 

27 

.23 

.16 

.23 

.38 

.23 

.00 

78 

52 

1.12 

.44 

.54 

.80 

.54 

.00 

78 

77 

2.37 

1.64 

1.75 

2.05 

1.37 

.00 

78 

125 

4.34 

3.29 

3.24 

3.38 

1.74 

.00 

78 

167 

6.05 

4.60 

4.52 

1.95 

.00 

78 

244 

7.55 

6.20 

5.25 

6.16 

2.26 

.00 

78 

341 

9.50 

7.50 

6.98 

2.50 

.00 

78 

421 

9.85 

8.05 

7.29 

2.70 

.00 

The  seven  different  varieties  of  mycoderma  yeasts,  whose  rates  of 
fermentation  in  normal  must,  concentrated  must,  beer  wort,  dextrose, 
saccharose  and  lactose  solutions  were  studied  gave  great  variation  in 
the  rate  of  fermentation  and  the  amounts  of  sugar  fermented.  Organ- 
ism 65  did  not  cause  fermentation  in  any  of  the  liquids  tested,  though 
it  gave  a  vigorous  growth.  On  the  other  hand,  yeasts  68,  73,  and  78, 
developed  fairly  strong  fermentations  in  must  and  beer  wort,  destroy- 
ing nearly  as  much  sugar  as  the  true  wine  yeasts.  Organism  76, 
caused  a  very  feeble  fermentation  in  grape  must  but  did  not  ferment 
any  of  the  other  liquids  tested.  The  mycoderma  forms  did  not  ferment 
so  rapidly  in  dextrose  solution  as  in  grape  must.  Contrasted  with  the 
mycoderma  forms  the  true  wine  yeasts  did  not  exhibit  this  difference. 
Lactose  was  not  fermented  by  any  of  the  mycoderma  forms.  Sac- 
charose was  attacked  to  a  slight  degree  by  forms  70,  73,  and  78,  the 
amount  of  sugar  actually  fermented  in  each  case  being  small.  In 
general,  the  fermentation  curves  of  the  forms  shown  in  the  diagrams 


28 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


differ  from  those  of  the  true  wine  yeasts  shown  in  figures  12  to  15,  by 
their  low  and  gentle  slope  throughout.  In  most  cases  they  show  a 
slower  start  of  fermentation  than  do  the  8.  ellipsoideus  yeasts.     Be- 


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40 

Fig.  8. — Fermentation  curves  of  Mycoderma  forms  in  must  of  2.3°  and  29 ?! 
Balling  and  beer  wort. 


cause  of  their  slow  and  incomplete  fermentations  the  mycoderma  forms 
discussed  above  are  not  desirable  types  to  have  in  wine  fermentations. 
Attenuation  and  Alcohol  Formation. — Analyses  were  made  of  the 
Liquids  fermented  by  the  mycoderma  forms  (table  4). 


1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


29 


TABLE  4 

Yeasts  Numbers  65,  68,  70,  71,  73,  76,  and  78.     Attenuation  and  Yields  of 

Alcohol  in  Must,  Beer  Wort,  Dextrose,  Saccharose,  and 

Lactose  Solutions 


feast 

No. 

Medium 

Attenuation 

in  grams 

per  100 

c.c 

Theoretical 
yield  of 
alcohol 

Observed 
yield  of 
alcohol 

Per  cent  of 

theoretical 

yield 

obtained 

64 

Must  of  23?  Balling  

0.00 

65 

Must  of  29!8  Balling  

0.00 
0.00 

65 

Beer  wort  of  14°  Balling  ... 

65 

Dextrose  solution,   15%  

0.00 

65 

Saccharose  solution,  15%   . 

0.00 

65 

Lactose  solution,  15%   

0.00 

68 

Must  of  23°  Balling  

...  19.88 

13.08 

7.45 

56.9 

68 

Must  of  32°  Balling  

...     7.84 

5.15 

4.50 

89.3 

68 

Beer  wort  of  14°  Balling  . 

...       .86 

.62 

.60 

96.8 

68 

Dextrose  solution,  15%   

....  12.68 

8.30 

6.40 

77.1 

68 

Saccharose  solution,   15%   . 

.03 

.02 

0.00 

00.0 

68 

Lactose  solution,  15%   

...       .27 

.19 

0.00 

00.0 

70 

Must  of  23°  Balling  

...     6.66 

4.38 

2.55 

58.2 

70 

Must  of  29?8  Balling  

....     2.05 

1.35 

1.00 

74.5 

70 

Beer  wort  of  14°  Balling  ... 

...     1.43 

.96 

.80 

83.3 

70 

Dextrose  solution,  15%  

...     4.51 

2.96 

1.70 

57.4 

70 

Saccharose  solution,   15%   . 

70 

Lactose  solution,  15%   

...     0.00 

0.00 

0.00 

00.0 

71 

Must  of  23°  Balling  

...  17.61 

11.58 

8.80 

75.9 

71 

Must  of  29?8  Balling  

7.90 

71 

Beer  wort  of  14°  Balling  ... 

...     2.91 

2.03 

0.00 

00.0 

71 

Dextrose  solution,  15%   .... 

....  12.20 

8.02 

4.10 

51.1 

71 

Saccharose  solution,  15%   . 

08 

.05 

0.00 

00.0 

71 

Lactose  solution,  15%  

16 

.11 

0.00 

00.0 

73 

Must  of  23°  Balling  

...  12.01 

7.90 

7.50 

94.9 

73 

Must  of  29?8  Balling  

...  14.75 

9.70 

6.80 

70.1 

73 

Beer  wort  of  14°  Balling  ... 

....     1.61 

1.16 

.52 

44.8 

73 

Dextrose  solution,   15%   .... 

....  12.60 

8.29 

5.60 

67.5 

73 

Saccharose  solution,  15%   . 

....     2.08 

1.50 

1.50 

100 

73 

Lactose  solution,  15% 

35 

.25 

0.00 

00.0 

76 

Must  of  23°  Balling  

0.00 
0.00 

76 

Must  of  29?8  Balling  

76 

Beer  wort  of  14°  Balling  ... 

0.00 

76 

Dextrose  solution,   15%   .... 

0.00 

76 

Saccharose  solution,   15%   . 

0.00 

76 

Lactose  solution,  15% 

0.00 

78 

Must  of  23°  Balling  

....  18.87 

12.41 

10.30 

83.1 

78 

Must  of  29?8  Balling  

....  14.13 

9.29 

9.00 

97.9 

78 

Beer  wort  of  14°  Balling  ... 

....  12.15 

7.90 

7.20 

91.1 

78 

Dextrose  solution,   15%   .... 

....  13.22 

8.69 

6.20 

71.3 

78 

Saccharose  solution,   15% 

....     3.93 

2.86 

1.40 

48.9 

78 

Lactose  solution,  15%  

36 

.26 

0.00 

00.0 

30  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

An  examination  of  the  data  given  in  table  4  shows  that  in  grape 
must,  which  was  the  most  favorable  medium  for  the  activity  of  the 
yeasts  under  observation,  amounts  of  alcohol  varying*  from  .00%  to 
10.3%  were  formed.  Some  of  the  organisms,  namely,  73  and  78,  gave 
good  yields  of  alcohol  for  the  sugar  fermented  in  grape  must,  while 
others  e.g.,  68,  70,  71,  gave  low  yields.  Yeasts  65  and  76  gave  no  alcohol 
in  any  of  the  liquids  in  which  they  were  grown,  although  they  destroyed 
small  amounts  of  sugar  in  some  of  the  solutions.  None  of  the  organisms 
of  this  group  fermented  beer  wort  to  any  notable  degree  and  the  fer- 
mentations that  did  take  place  in  this  liquid  probably  represented  the 
destruction  of  dextrose  present,  only,  while  it  is  probable  that  the  mal- 
tose was  not  attacked.  Saccharose  likewise  proved  very  resistant  to  fer- 
mentation by  the  above  forms.  Organisms  68,  71,  73,  and  78  formed 
considerable  amounts  of  alcohol  in  dextrose  solution,  indicating  that 
this  sugar  is  more  easily  attacked  than  cane  sugar  or  maltose.  These 
facts  indicate  that  the  yeasts  under  discussion  are  deficient  in  invertase 
and  maltase,  enzymes  that  are  necessary  to  the  fermentation  of  saccha- 
rose and  maltose. 

Since  the  mycoderma  forms  discussed  above  do  not  ferment  beer 
wort  or  cane  sugar,  they  would  be  useless  in  the  manufacture  of  beer 
or  of  alcohol  from  materials  containing  cane  sugar.  The  amounts  of 
alcohol  formed  in  grape  must  by  several  of  the  organisms  might  be 
sufficient  for  some  purposes,  but  their  use  for  the  production  of  wine 
is  excluded  by  the  fact  that  all  of  them  produce  disagreeable  tastes  and 
odors  in  grape  must,  resulting  in  the  formation  of  an  undririkable 
fermented  liquid  in  all  cases.  Therefore,  it  is  to  the  winemaker's 
interest  to  prevent  the  growth  of  these  organisms. 

TOEULA  FORMS  FROM  CALIFORNIA  GRAPES 

Two  yeasts  of  the  torula  type  were  found  on  grapes  examined 
in  1911,  one  from  Davis  grapes  and  the  other  from  grapes  grown  in 
Contra  Costa  County,  California. 

Organism  37.    Torula  Yeast  from  Grapes  Grown  at  Davis 

Morphology. — Microscopical  Appearance. — Varies  from  spherical 
to  shortly  ellipsoidal  in  form;  latter  form  predominating.  Cells  tend 
to  form  chains  of  cells  of  various  sizes  (see  fig.  3). 

Size. — Average,  4.6/x  x  3.7/x.  Maximum,  15/x  x  6fi.  Minimum,  3/x  x  3/^. 

Spore  Formation. — No  spores  formed  at  22°  C  or  28°  C. 


1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


31 


Colonies  on  Agar  Agar. — Almost  flat,  but  centers,  slightly  elevated. 
White.  Waxy  luster.  Edges  entire.  Colonies  in  the  agar,  spherical. 
Under  microscope,  edges  of  colonies  entire. 

Colonies  on  Gelatin. — Small.  White.  Waxy  luster.  Gelatin  quickly 
liquefied. 

Growth  in  Liquid  Media. — Growth  in  grape  must,  dextrose,  and  beer 
wort,  rapid,  with  formation  of  considerable  sediment,  and  develop- 
ment of  a  feeble  bottom  fermentation.  No  film  formation.  No  fer- 
mentation in  lactose  solutions,  but  good  growth.  Feeble  fermenta- 
tion and  good  growth  in  saccharose.  Rancid  taste  and  odor  developed 
in  fermented  liquids. 


40     no      izo    if>o   znn    2.-40  2.?>n   ^zo  ?*>f>c>  ioo  4*0 

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Fig.  9. — Fermentation  curves  of  Mycoderma  forms  in  dextrose  and  saccha- 
rose solutions. 


Organism  77.     Torula  from  Contra  Costa  County  Grapes 

Morphology. — Microscopical  Appearance. — Vary  from  ellipsoidal 
to  sausage-shaped  with  ellipsoidal  form  predominating  (see  fig.  3). 
Tends  to  form  short  chains. 

Size. — Average,  4/x,  x  2.1  fx.  Maximum,  8p  x  4.5^.  Minimum, 
2.2/*xl.5/x. 

Colonies  on  Agar  Agar. — Small.  White.  Convex.  Waxy  luster. 
Spherical  colonies  in  the  agar.  Under  the  microscope  colonies  are 
found  to  be  surrounded  by  straight  chains  of  long  cells.  Most  other 
yeasts  examined  that  formed  side  chains  in  this  manner  produced 
branching  cells. 

Colonies  on  Gelatin. — Contoured  surface  with  highest  elevation  at 
centers  of  colonies.    Centers  of  colonies  colorless.     Chalklike  at  edges. 


32  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Undermicroscope,  colonies  are  fringed  with  branching  chains  of  long 
cells. 

Growth  in  Liquid  Media. — Growth  in  all  liquids  slow.  Feeble  fer- 
mentation in  grape  must,  beer  wort,  and  dextose.  No  surface  growth. 
No  fermentation  in  lactose  and  saccharose. 

Rates  of  Fermentation  of  Torulas  37  and  77  in  Various  Media. 

Kates  of  fermentation  in  grape  must  of  23°  Balling,  and  29.8°  Balling, 
beer  wort  of  14°  Balling,  and  15%  solutions  of  dextrose,  saccharose, 
and  lactose  were  tested  as  previously  described  for  apiculatus  and 
mycoderma  forms.    The  results  are  given  in  table  5  and  figure  10. 

TABLE  5 

Fermentation  Tests  at  33°  C  op  Torula  Forms  from  California  Yfasts 

Must  of  Dextrose      Saccharose       Lactose 

Must  of  of29°8         Wort  loss,  loss  in  loss,  in  loss,  in 

13°  Balling,     Balling,         in  grams  grams  grams  grams 


Yeast 
number 

Time  in 
hours 

loss  in 
grams 

loss,  in 
grams 

14  per  cent 
solution 

15  per  cent 
solution 

15  per  cent 
solution 

15  per  ci 
solutio 

37 

18 

.01 

,00 

.04 

.12 

.07 

.02 

37 

42 

.19 

.07 

.31 

.54 

.29 

.07 

37 

66 

.51 

.45 

.98 

.38 

37 

92 

.63 

.50 

1.40 

.43 

.08 

37 

165 

.87 

.51 

2.74 

.54 

.08 

37 

236 

.87 

.13 

.51 

3.64 

.54 

.09 

77 

46 

.17 

.01 

.23 

.23 

.01 

.03 

77 

73 

.84 

.15 

.48 

.48 

77 

96 

1.14 

.42 

.58 

.58 

.10 

77 

118 

1.30 

.81 

.68 

.68 

77 

167 

1.37 

.86 

.78 

.78 

.10 

77 

215 

1.44 

1.03 

.83 

.83 

77 

239 

1.48 

1.08 

.95 

.95 

.03 

Torulas  37  and  77  were  able  to  produce  only  very  feeble  fermenta- 
tions in  the  liquids  tested.  Torula  yeast  77  was  not  able  to  ferment 
must  of  30°  Balling,  or  saccharose  solution.  In  must  of  23°  Balling, 
and  in  beer  wort  the  fermentations  of  the  two  yeasts  were  about  equal. 
In  must,  beer  wort,  and  saccharose  the  fermentations  were  completed 
in  a  short  time,  about  120  hours.  In  comparison  with  the  fermenta- 
tion curves  of  the  8.  elUpsoideus  yeasts  (figs.  12  to  15),  the  yeasts 
above  gave  very  weak  and  incomplete  fermentations.  The  flavors 
of  the  fermented  liquids  were  disagreeable.  Therefore,  the  yeasts 
seen i  to  have  no  practical  value  but  may  cause  defective  flavors  in 
wine  where  their  growth  is  not  prevented. 

Attenuation  and  Yields  of  Alcohol. — The  fermented  liquids  were 
analyzed  for  alcohol  with  the  results  reported  in  table  6. 


1918] 


Cruess:    Fermentation  Organisms  of  California  Grapes 


33 


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Fig.  10. — Fermentation  curves  of  yeasts  37  and  77  in  grape  must,  beer  wort, 
dextrose  and  saccharose  solutions. 


34  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

TABLE  6 

Yeasts  37  and  77.    Attenuation  and  Yields  of  Alcohol  in  Must,  Beer  Wort, 
Dextrose,  Saccharose,  and  Lactose  Solutions 

Attenuation  Per  cent  of 

in  grams  Theoretical  Observed  theoretical 

Yeast                                                                        per  100  yield  of            yield  of  yield 

No.                            Medium                               c.c.  alcohol              alcohol  obtained 

37  Must  of  23°  Balling  5.21  3.42  .60  17.6 

37  Must  of  29?8  Balling  05  .03  .00  .0 

37  Beer  wort  of  14°  Balling  .20 

37  Dextrose  solution,  15%   7.96  5.22  3.50  67.3 

37  Saccharose  solution,  15% 64  .43  .40  93.02 

37  Lactose  solution,  15%   06  .04  .00  .0 

77  Must  of  23°  Balling  4.58  2.99  1.31  43.7 

77  Must  of  29?8  Balling  3.85  2.53  .65  25.7 

77  Beer  wort  of  14°  Balling  ....     2.91  2.00  .30  15.0 

77  Dextrose  solution,  15%   4.55  2.99  .65  21.7 

77  Saccharose  solution,   15%   .00  .0 

77  Lactose  solution,  15%   06  .04  .00  .0 

Analyses  of  the  fermented  liquids  from  fermentation  of  Torulas 
37  and  77  in  grape  must,  etc.,  showed  that  the  absolute  amounts  of 
alcohol  formed  were  small  and  that  the  yields  for  each  per  cent  of 
sugar  destroyed  were  in  all  cases,  except  for  yeast  37  in  dextrose,  very 
low.  In  the  majority  of  the  fermentations,  the  yields  were  less  than 
50  per  cent  of  the  theoretical.  These  yeasts  have  the  defects  of  giving 
slow  incomplete  fermentations,  low  yields  of  alcohol  for  sugar  fer- 
mented and  of  producing  liquids  of  disagreeable  odor  and  flavor. 


PASTORIANUS  AND  WILLI  A  YEAST  FROM  CALIFORNIA  GRAPES 

One  S.  pastorianus  and  one  Willia  yeast  were  obtained  from  grapes 
in  1911.    Both  are  true  yeasts  and  form  spores. 

Organism  36.    S.  pastorianus  Yeast  from  Grapes  Grown  at  Davis 

Morphology.  —  Microscopical  Appearance.  —  Form  varies  from 
spherical  to  sausage-shaped.  The  ellipsoidal  form  predominating. 
Much  smaller  than  true  ellipsoideus  yeast  (fig.  3). 

Size. — Average,  4.5/*  x  3/x.  Maximum,  7.5w  x  3/x.  Minimum, 
1.5/x  x  1.5ft. 

Spore. — Produced  in  great  abundance  in  24  hours  on  gypsum  spore 
blocks.  Spores  also  form  in  grape  must  cultures.  Spores  spherical  in 
form.    Number  per  cell,  2  to  4.    Size  of  spores,  2/x  x  2/x,  (fig.  7) . 

Colonies  on  Agar  Agar. — Round.  Slightly  convex.  White.  "Waxy 
Luster.    Colonies  in  agar  spherical.    Under  the  low  power,  the  colonies 


1918]  Cruess :   Fermentation  Organisms  of  California  Grapes  35 

are  surrounded  by  chains  of  cells  made  up  of  long  central  cells  with 
pairs  of  short  ellipsoidal  cells  at  the  junctures  of  the  larger  cells. 

Colonies  on  Gelatin. — Flat.  Concentric  rings  of  growth.  Outer 
edges  of  colonies  sharply  indented.  Gelatin  slowly  softened  but  not 
completely  liquefied. 

Growth  in  Liquid  Media. — Heavy  wrinkled  surface  growth  which 
sinks  to  bottom  frequently.  No  visible  fermentation.  Taste  of  liquids 
bitter  and  disagreeable. 

Organism  69.    Willia  Species  (Saccharomyces  anomalus)  from  Grapes 
Grown  at  Acampo,  California 

Morphology. — Microscopical  Appearance. — Size  and  shape  very 
irregular.  The  larger  cells  usually  sausage-shaped  or  ellipsoidal  and 
the  smaller  ones  spherical.  Larger  than  typical  S.  anomalus  in  the 
University  collection  (fig.  3). 

Size. — Average,  6/a  x  3^.   Maximum,  14^  x  2.5//,.   Minimum,  3/x  x  3/x. 

Spore  Formation. — Spores  are  formed  abundantly  on  gysum  blocks 
at  28°  C  and  22°  C,  in  three  days.  Spores  are  of  typical  Willia  form, 
i.e.,  hat-shaped  (fig.  7).  Number  of  spores  per  cell  varies  from  2  to  8. 
Size  of  spores,  4//,  x  1.5/x. 

Colonies  on  Agar  Agar. — Medium  size.  Flat.  White.  Waxy 
luster.  Edges  entire.  Colonies  on  the  agar  are  spherical.  LTnder  the 
microscope  the  edges  of  the  colonies  are  entire. 

Colonies  on  Gelatin. — Large.  Flat.  White.  Chalklike.  Colonies 
spread  rapidly  and  soon  cover  entire  surface  and  the  gelatin  is  rapidly 
liquefied. 

Growth  in  Liquid  Media. — A  chalky  white  film  develops  on  all 
liquids  tested,  with  flocculent  growth  at  bottom  of  flask.  Prolonged 
top  fermentation  takes  place  in  all  liquids  except  lactose.  Strong 
odor  of  ethyl  acetate  is  developed.  The  fermented  liquids  possess  a 
burning  taste. 

Rates  of  Fermentation  of  Yeasts  36  and  69. — These  yeasts  were 
tested  for  their  rates  of  fermentation  in  various  media  as  previously 
described  for  other  organisms.  The  results  are  given  in  table  7  and 
figure  11. 

From  the  data  given  in  table  7,  yeast  36  is  seen  to  give  scarcely  any 
fermentation  in  any  of  the  liquids  tested,  the  maximum  loss  in  weight 
per  100  c.c.  being  .68  grams  in  must  of  29.8°  Balling.  On  the  other 
hand,  yeast  69  gave  fairly  strong  fermentations  in  must,  beer  wort, 
dextrose,  and  saccharose  solutions.     Neither  yeast  fermented  lactose, 


36  University  of  California  Publications  in  Agricultural  Sciences  '      [Vol.  4 

TABLE  7 
Data  of  Eates  of  Fermentation  of  Yeasts  Numbers  36  and  69  in  Various 

Sugar  Solutions 


Yeast 
number 

Time  in 
hours 

Must  of 

23°  Balling, 

loss  in 

grams 

Concentrated  Wort  loss, 
must,  loss       in  grams 
in  grams      14  per  cent 
29? 8  Ball.       Balling. 

loss,  in 

grams 

15  per  cent 

solution 

loss,  in 

grams 

15  per  cent 

solution 

loss,  in 

grams 

15  per  cer 

solution 

36 

46 

.03 

.03 

.03 

.03 

.00 

.00 

36 

73 

.04 

.04 

.03 

.06 

.00 

.00 

36 

96 

.09 

.11 

.03 

.08 

.00 

.01 

36 

118 

.24 

.16 

.13 

.13 

.00 

.01 

36 

167 

.33 

.43 

.13 

.19 

.00 

.02 

36 

215 

.45 

.63 

.13 

.26 

.00 

.02 

36 

239 

.46 

.68 

.28 

.00 

.02 

69 

18 

.01 

.10 

.13 

.01 

.10 

.03 

69 

42 

.58 

.18 

.46 

.13 

.28 

.14 

69 

66 

1.10 

.23 

.56 

.77 

.86 

.14 

69 

92 

1.79 

.80 

1.71 

1.77 

1.90 

69 

123 

2.49 

2.20 

1.75 

2.87 

2.88 

69 

165 

3.22 

2.50 

1.81 

3.99 

3.78 

69 

236 

4.02 

4.88 

1.92 

5.31 

4.62 

.14 

69 

283 

4.49 

5.55 

1.93 

5.77 

4.98 

69 

314 

4.62 

5.75 

5.79 

5.03 

while  yeast  36  did  not  attack  saccharose.  Although  yeast  69  gives 
more  vigorous  fermentations  than  does  yeast  36,  still  it  does  not  ap- 
proach the  S.  ellipsoideus  yeasts  from  California  grapes  in  rate  of 
fermentation  or  fermentative  power. 

Attenuation  and  Alcohol  Yields  of  Yeasts  36  and  69. — The  fer- 
mented liquids  were  analyzed  with  the  results  given  in  the  accom- 
panying table. 

TABLE  8 

Yeasts  Numbers  36  and  69.     Attenuation  and  Yields  of  Alcohol  in  Must, 

Beer  Wort,  Dextrose,  Saccharose,  and  Lactose  Solutions 

Attenuation  Per  cent  of 

in  grams  Theoretical  Ons€rved  theoretical 

Yeast                                                                         per  100  yield  of  yield  of  yield 

No.                             Medium                                c.c  alcohol  alcohol  obtained 

36  Must  of  23°  Balling  2.49  1.95  .30  15.3 

36  Must  of  29?8  Balling  1.65  1.08  .00  .0 

36  Beer  wort  of  14°  Balling 31  .20  .00  .0 

36  Dextrose  solution,  15%  1.95  1.28  .24  18.9 

36  Saccharose  solution,  15% 02  .67  .00 

36  Lactose  solution,  15%   11  .07  .00 

69  Must  of  23°  Balling  11.12  7.30  1.25  17.1 

69  Must  of  29?8  Balling  2.92  1.90  1.00  52.6 

69  Beer  wort  of  14°   Balling  ....     1.40  .92  .50  54.3 

69  Dextrose  solution,  15%   11.03  7.25  3.10  42.6 

69  Saccharose  solution,  15%  7.73  5.09  2.60  51.2 

69  Lactose  solution,  15%   1.25  .80  .00  .0 


1918] 


Crusss:   Fermentation  Organisms  of  California  Grapes 


37 


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Fig.  11. — Fermentation   curves   for  yeasts   36   and   69   in   grape   must,   beer 
wort,  dextrose,  and  saccharose  solution  at  33  °C. 


38  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Yeast  69  destroyed  larger  amounts  of  sugar  than  did  yeast  36  and 
formed  greater  amounts  of  alcohol,  but  both  yeasts  gave  very  low 
yields  of  alcohol  compared  to  the  amount  of  sugar  fermented.  In 
most  cases  the  yield  was  below  50%  of  the  theoretical,  based  on  the 
sugar  destroyed.  Yeast  69  formed  3.1%  alcohol  in  dextrose  solution, 
this  being  the  largest  amount  of  alcohol  produced  by  either  yeast. 

Because  of  its  disagreeable  flavor  and  low  alcohol  formation,  yeast 
36  is  worthless  for  fermentation  purposes.  Yeast  69  produces  a  very 
aromatic  odor  and  flavor  in  liquids  in  which  it  grows.  These  fer- 
mented materials  are  not  pleasing  to  drink  by  themselves  but  when 
added  in  small  amounts  to  new  wine  they  have  the  property  of 
"aging"  the  new  wine  so  treated.  Therefore  it  may  be  possible  to  use 
this  yeast  as  an  aid  to  aging  wine  either  by  adding  some  of  the  liquid 
fermented  by  it  to  new  wine  or  by  growing  it  directly  in  new  wine, 
and  in  this  latter  case,  allowing  the  desired  flavors  to  be  developed  in 
the  wine  itself.  Yeasts  of  this  same  group  are  used  to  some  extent 
in  the  aging  of  "sake"   (Japanese  beer). 

TEUE  WINE  YEASTS,  SACCHAROMYCES  ELLIPSOIDEUS  FROM 
CALIFORNIA  GRAPES 

Six  kinds  of  true  wine  yeast,  S.  ellipsoideus,  were  isolated  from 
grapes  in  1911  and  have  been  discussed  as  a  group  in  the  following 
pages. 

Organism  64.    Saccharomyces  ellipsoideus  from  Grapes 
Grown  at  Acampo 

Morphology.  —  Microscopical  Appearance.  —  Form  varies  from 
shortly  ellipsoidal  to  spherical.  Cells  shorter  than  those  of  no.  74 
(fig.  5). 

Size. — Average,  7.5/x,  x  6/x.   Maximum,  12//,  x  3/x.   Minimum,  3/x  x  3/x. 

Spore  Formation. — Abundant  spore  formation  at  22°  C  and  28°  C. 
Usually  3  spores  per  cell.     Shape  spherical.     Size,  3,/x  x  3/x. 

Colonies  on  Agar  Agar. — Medium  size.  Round.  Moderately  con- 
vex. White.  Waxy  luster.  Edges  entire.  Under  microscope  edges 
are  entire. 

Colonies  on  Gelatin. — Large.  Centers  elevated.  Radiating  ridges 
from  center.  Centers  slightly  elevated.  Edges  of  colonies  are  un- 
dulate in  appearance.  Gelatin  slowly  softened  but  not  liquefied. 
Edges  of  colonies  entire  under  microscope. 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  39 

Growth  in  Liquid  Media. — Rapid  in  grape  must,  beer  wort,  dex- 
trose, and  saccharose  solutions  with  vigorous  fermentation  and  forma- 
tion of  fine  grained  sediment.  No  fermentation  in  lactose,  but  fair 
growth.     Characteristic  vinous  odor  and  flavor  developed. 

Organism  66.     Saccharomyces  ellipsoideus  from  Grapes 
Grown  at  Davis 

Morphology. — Microscopical  Appearance.  —  Predominating  form 
oblong  with  tendency  to  be  pointed  at  one  end.  Resembles  S.  cerevisiae 
in  this  regard  (fig.  5). 

Size. — Average,  9/x,  x  5.7/x.  Maximum,  13.5//,  x  4/*.  Minimum, 
4/x  x  3/x.  Considerably  larger  than  most  varieties  of  S.  ellipsoideus 
from  California  grapes. 

Spore  Formation. — Spores  are  formed  abundantly  at  22°  C  and 
28°  C  but  more  rapidly  at  the  lower  temperature.  Number  per  cell 
2  to  3.     Size,  3/*  x  3/*. 

Colonies  on  Agar  Agar. — Size,  medium.  Convex.  Circular. 
White.  Waxy  luster.  Edges  entire  macroscopically  and  microsco- 
pically.    Colonies  in  the  agar  are  usually  lens-shaped. 

Colonies  on  Gelatin. — Colonies  more  flat  than  on  agar.  Smooth. 
Under  microscope  edges  of  colonies  appear  bordered  with  branching 
chains  of  ellipsoidal  cells.     Gelatin  slowly  softened  but  not  liquefied. 

Growth  in  Liquid  Media. — Rapid  with  vigorous  fermentation  in  all 
solutions  tested  except  lactose.  Grows  in  lactose.  Sediment  fine 
grained.  Settles  quickly  after  fermentation.  Typical  vinous  flavor 
developed  in  fermented  liquids. 

Organism  67.     Saccharomyces  ellipsoideus  from  Grapes  Grown 
in  Contra  Costa  County 

Morphology. — Microscopical  Appearance. — Ellipsoidal  to  spherical, 
former  type  predominating.  Resembles  yeast  74  in  general  appear- 
ance (fig.  5). 

Size. — Average,  6.75/x  x  5.25/x.  Maximum,  9jx  x  6.75/x.  Minimum, 
3/x  x  3/x. 

Spore  Formation. — Large  numbers  of  spores  are  formed  at  22°  C 
and  28°  C  in  24  hours  on  spore  blocks.  Number  per  cell,  2  or  3.  Size, 
3/x  x  3/x. 

Colonies  on  Agar  Agar. — Small.  Convex.  White.  Waxy  luster. 
Colonies  in  agar  are  spherical.  Edges  of  colonies  entire,  both  macro- 
scopically and  microscopically. 


40  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Colonies  on  Gelatin. — Large.  Centers  of  colonies  slightly  de- 
pressed. Edges  are  undulate.  Under  microscope,  colonies  are  fringed 
with  short  straight  chains  of  ellipsoidal  cells.  The  gelatin  is  not 
liquefied  or  softened. 

Growth  in  Liquid  Media. — Growth  rapid  and  fermentation  vigor- 
ous in  grape  must,  beer  wort,  dextrose,  and  saccharose.  Growth  slight 
and  fermentation  absent  in  lactose.  Fermentation  more  rapid  at  20°  C 
than  at  33°  C.     Sediment  fine  grained.     Typical  vinous  flavor  and 

Organism  74.    Saccharomyces  ellipsoideus  from  Grapes  Grown 

at  Acampo 

Morphology. — Microscopical  Appearance.  —  Usual  form  oblong 
rather  than  shortly  ellipsoidal.  Decidedly  different  in  form  than 
yeast  64  from  the  same  grapes  (fig.  5). 

Size. — Average,  6.6/x  x  4.6^.  Maximum,  9/x,  x  4.5/x.  Minimum, 
Sfi  x  3/x.    Smaller  than  yeasts  66  and  79,  which  it  resembles  in  shape. 

Spore  Formation. — Few  spores  only  at  28°  C  in  48  hours  and  none 
in  24  hours.  More  formed  at  22°  C  in  48  hours.  Number  per  cell,  2. 
Size,  Sjx  x  3/x  (fig.  6). 

Colonies  on  Agar  Agar.  —  Large.  Round.  Centers  elevated. 
Colonies  in  agar  lens-shaped.  Edges  of  colonies  entire  both  macrosco- 
pically  and  microscopically. 

Colonies  on  Gelatin. — Large.  Low  elevation.  Colonies  in  the 
gelatin  spherical.  Edges  of  colonies  entire  both  macroscopically  and 
microscopically.     No  liquefaction. 

Growth  in  Liquid  Media. — Rapid  with  vigorous  fermentation  in 
must,  beer  wort,  dextrose,  and  saccharose  solutions.  Small  growth  and 
no  fermentation  in  lactose.    Typical  vinous  taste  and  odor. 

Organism  75.    Saccharomyces  ellipsoideus  from  Grapes  Grown  in 
Contra  Costa  Cownty 

Morphology. — Microscopical  Appearance.  —  Spherical  to  shortly 
ellipsoidal  (fig.  5). 

Size. — Average,  6.3//.  x  5.5/x.  Maximum,  8^  x  8/x.  Minimum,  3^  x  3fx. 

Spore  Formation. — No  spores  formed  at  28°  C  at  72  hours,  but 
abundantly  after  144  hours.  This  differs  from  yeast  67  from  the  same 
grapes;  67  forms  spores  in  24  hours  at  28°  C.  Numbers  of  spores  per 
cell,  2.    Size,  3^x3^  (fig.  6). 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  41 

Colonies  on  Agar  Agar. — Medium  size.  Round.  Convex.  White. 
Waxy  luster.  Colonies  in  the  agar  are  lens-shaped.  Edges  of  colonies 
entire,  both  macroseopieally  and  microscopically. 

Colonies  on  Gelatin. — Large.  Low  and  of  even  elevation.  Sur- 
face radiately  ridged.  Edges  undulate.  Under  microscope  colonies 
are  bordered  by  short  straight  chains  of  ellipsoidal  cells. 

Growth  in  Liquid  Media. — Rapid  and  fermentation  vigorous  in 
grape  must,  beer  wort,  dextrose,  and  saccharose.  Slight  and  no  fer- 
mentation in  lactose.  Typical  vinous  flavor  and  odor  in  fermented 
liquids. 

Organism  79.    Saccharomyces  ellipsoideus  from  Grapes  Grown  in 
Contra  Costa  County 

Morphology. — Microscopical  Appearance. — Cells  large  and  oblong. 
Not  strictly  ellipsoidal  in  form  (fig.  6). 

Size. — Average,  8.25/x  x  5/x.  Maximum,  12/*  x  6/x.  Minimum, 
4.5/a  x  3/a. 

Spore  Formation. — Spores  are  formed  abundantly  in  24  hours  at 
28°  C.    Number  per  cell,  2  to  4.    Size,  2/i  x  2/*  and  3/x  x  3/x  (fig.  6) . 

Colonies  on  Agar  Agar. — Medium  size.  Spherical.  Convex.  White. 
Waxy  luster.  Colonies  in  agar  lens-shaped.  Edges  entire  both  macro- 
seopieally and  microscopically. 

Colonies  on  Gelatin.  Flat.  Edges  hazy.  White.  Waxy  luster. 
Under  microscope  edges  are  bordered  by  branching  chains  of  el- 
lipsoidal cells. 

Growth  in  Liquid  Media. — In  grape  must,  beer  wort,  dextrose,  and 
saccharose,  rapid  and  fermentation  vigorous.  In  lactose,  slow  and  no 
fermentation.    Typical  vinous  taste  and  odor  developed. 

Fermentation  Records  at  33°  C. — Fermentations  were  carried  out 
in  100  c.c.  portions  of  various  liquids  at  33°  C  and  records  of  the  rates 
of  fermentation  were  kept  as  previously  explained. 

All  of  the  yeasts  with  the  exception  of  yeast  67  show  rapid  fer- 
mentations in  must  of  23°  Balling,  and  30°  Balling,  beer  wort,  dextrose 
and  saccharose  solutions,  there  being  little  to  choose  between  them 
in  the  rapidity  of  fermentation  and  the  amount  of  sugar  destroyed. 
The  yeasts  show  considerable  variation  in  the  fermentation  of  beer 
wort.  Apparently  some  of  the  yeasts  ferment  only  the  dextrose  of 
the  beer  wort  and  not  the  maltose.  Yeast  67,  however,  is  very  strongly 
inhibited  in  its  activity  by  the  moderate  temperature  of  33°  C,  because 
it  causes  only  very  feeble  and  incomplete  fermentations  at  this  tern- 


42 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


TABLE  9 

Data  on  Eates  of  Fermentation  of  Yeasts  Numbers  64,  66,  67,  74,  75,  and  79 

at  33°  C  in  Various  Sugar  Solutions 


Yeast 
number 

64 

64 

64 

64 

64 

64 

64 


79 
79 
79 
79 
79 
79 
79 


Time  in 
hours 

18 

42 

66 

92 
123 
165 
236 


46 
73 
96 
118 
167 
215 
239 


Must  of 

23°  Balling, 

loss  in 

grams 

1.42 

4.18 
6.72 
8.09 
8.26 
8.33 
8.88 


Must  of 
of  29?8 
Balling 
loss  in 
grams 

.00 

.66 
4.33 
7.90 
8.70 
9.70 
10.00 


Wort  loss, 
in  grams 
14°  Ball. 

.87 

2.57 

2.75 

2.94 

2.94 

3.04 

3.16 


Dextrose      Saccharose 
loss,  in  loss  in 

grams  grams 

15  per  cent  15  per  cent 
solution 


.49 
1.95 
2.32 

4.74 
5.84 
6.49 
6.77 


4.96 

7.77 
8.40 
8.74 
8.88 
9.07 
9.10 


3.21 
6.97 
8.84 
10.14 
10.83 
11.27 
11.40 


1.81 
2.42 
2.63 

2.83 
2.83 
3.07 
3.07 


1.79 
3.37 
4.52 
5.66 
6.38 
6.66 
6.68 


solution 

.68 
2.43 
3.96 
5.35 
6.30 
6.88 
7.30 


1.80 
3.66 
4.77 
5.71 
6.40 
6.55 
6.60 


Lactose 

loss,  in 

grams 

15  per  cent 

solution 

.03 


.07 
.07 


66 

27 

1.83 

.05 

.06 

.44 

.60 

.00 

66 

52 

5.92 

.69 

.55 

2.13 

1.89 

.00 

66 

77 

8.49 

4.11 

2.49 

5.23 

2.91 

.00 

66 

125 

10.09 

9.31 

6.64 

6.81 

4.59 

.00 

66 

167 

10.44 

11.71 

8.69 

5.56 

.00 

66 

244 

10.98 

11.85 

7.54 

7.38 

.00 

66 

341 

11.68 

12.59 

8.35 

.00 

66 

421 

12.82 

8.55 

.00 

67 

27 

.14 

.04 

.33 

.66 

.31 

.00 

67 

52 

.89 

.14 

.53 

1.80 

.42 

.00 

67 

77 

1.99 

.92 

.64 

3.21 

.64 

.00 

67 

125 

2.79 

1.57 

.67 

4.11 

.83 

.00 

67 

244 

2.99 

1.84 

.72 

5.72 

.84 

.00 

67 

341 

3.33 

2.40 

.75 

7.07 

1.13 

.00 

67 

421 

3.50 

2.85 

.75 

7.35 

1.30 

.00 

67 

4.59 

2.98 

1.39 

.00 

74 

27 

2.69 

.16 

1.76 

.83 

.93 

.00 

74 

52 

5.71 

2.89 

2.54 

3.52 

2.74 

.00 

74 

77 

7.81 

6.15 

3.99 

5.52 

4.42 

.00 

74 

125 

9.14 

9.77 

4.27 

6.59 

.00 

74 

167 

9.31 

11.87 

4.32 

7.23 

7.09 

.00 

74 

244 

10.87 

12.38 

4.65 

7.62 

.00 

74 

341 

11.50 

12.53 

5.00 

7.87 

.00 

74 

421 

12.65 

5.15 

7.97 

.00 

75 

27 

2.08 

.08 

.66 

.38 

.61 

.00 

75 

52 

4.72 

.67 

1.29 

.80 

1.57 

.00 

75 

77 

6.74 

4.05 

1.89 

2.05 

2.47 

.00 

75 

125 

8.06 

6.89 

2.64 

3.38 

4.04 

.00 

75 

167 

8.74 

7.94 

3.12 

6.17 

5.17 

.00 

75 

244 

9.85 

8.88 

3.40 

6.98 

6.78 

.00 

75 

341 

10.97 

10.66 

3.55 

7.29 

7.60 

.00 

75 

421 

11.35 

10.66 

3.65 

7.90 

.00 
.00 
.00 
.00 
.00 
.00 
.00 


1918] 


Cruess:   Fermentation  Organisms  of  California  Grapes 


43 


perature.  The  presence  of  such  yeast  as  this  in  natural  wine  fermen- 
tations would  render  such  fermentations  very  liable  to  "sticking"  if 
the  temperature  arose  much  above   33°  C.     It  is  possible  that  the 


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Fig.  12. — Fermentation  curves  of  true  wine  yeasts  from  California  grapes. 

reason  that  some  wines  will  ferment  completely  even  at  temperatures 
above  38°  C,  and  others  stick  with  unfermented  sugar  is  because  of 
the  difference  in  the  character  of  the  natural  wine  yeast  present.  The 
possibility  of  a  yeast  similar  to  yeast  67  being  the  only  wine  yeast 
present  in  a  natural  wine  fermentation  makes  it  very  desirable  to 


44  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

insure  good  fermentations  by  the  use  of  pure  and  selected  varieties 
of  wine  yeast. 

Lactose  supported  the  growth  of  the  8.  ellipsoideus  yeasts  discussed 
above,  but  was  not  fermented  by  any  of  them. 

Attenuation  and  Alcohol  Formation  at  33°  C. — The  fermented 
liquids  were  analyzed  with  the  results  in  table  10. 

TABLE  10 

Yeasts  Numbers  64,  66,  67,  74,  75,  79.    Attenuation  and  Yields  of  Alcohol 
in  Must,  Beer  Wort,  Dextrose,  Saccharose,  and  Lactose  Solutions 


Sfeasl 
No. 

Medium 

Attenuation 
in  grams 
per  100 
c.c 

Theoretical 
yield  of 
alcohol 

Observed 
yield  of 
alcohol 

Per  cent  of 

theoretical 

yield 

obtained 

64 

Must  of  23°  Balling  

...  19.70 

12.80 

11.75 

93.4 

64 

Must  of  29?8  Balling  

...  21.40 

13.90 

12.80 

92.1 

64 

Beer  wort  of  14°  Balling  ... 

...     6.16 

4.39 

3.40 

77.4 

64 

Dextrose  solution,  15%  

...  11.69 

7.60 

6.00 

79.0 

64 

Saccharose  solution,  15%  ... 

...  12.23 

8.80 

7.15 

81.4 

64 

Lactose  solution,  15%  ... 

.00 

.0 

66 

Must  of  23°  Balling  

...  19.41 

12.70 
12.90 

11.40 
11.40 

89.7 

66 

Must  of  29?8  Balling  

...  19.75 

88.4 

66 

Beer  wort,  of  14°  Balling  . 

5.70 

66 

Dextrose  solution,  15%  

...  13.20 

8.60 

7.95 

92.4 

66 

Saccharose  solution,  15%  ... 

...  12.03 

8.30 

7.55 

90.9 

66 

Lactose  solution,  15%  

.00 

67 

Must  of  23°  Balling  

...     5.41 

3.55 

2.00 

56.4 

67 

Must  of  29?8  Balling  

...     3.85 

2.53 

1.30 

51.3 

67 

Beer  wort  of  14°  Balling  . 

...     1.61 

1.11 

.65 

58.6 

67 

Dextrose  solution,  15%  

...  12.75 

8.38 

7.95 

94.7 

67 

Saccharose  solution,  15%  ... 

...     2.82 

1.94 

1.00 

51.5 

67 

Lactose  solution,  15%   

...       .36 

.24 

.00 

.0 

74 

Must  of  23°  Balling  

...  19.31 

12.70 

11.40 

89.7 

74 

Must  of  29?8  Balling  

...  20.95 

13.70 

11.40 

83.2 

74 

Beer  wort,  of  14°  Balling  . 

...     7.96 

5.30 

4.10 

75.3 

74 

Dextrose  solution,  15%   

...  11.64 

7.60 

5.70 

75.0 

74 

Saccharose  solution,  15%  ... 

...  11.83 

8.18 

7.95 

97.2 

74 

Lactose  solution,  15%  

.00 

75 

Must  of  23°  Balling  

...  18.31 

12.03 

8.01 

66.6 

75 

Must  of  29?8  Balling  

...  22.10 

14.53 

75 

Beer  wort  of  14°  Balling  . 

...     6.07 

4.17 

2.34 

57.3 

75 

Dextrose  solution,   15%  

...  12.55 

8.60 

6.40 

74.4 

75 

Saccharose  solution,  15%  ... 

...  12.62 

8.70 

8.60 

98.8 

75 

Lactose  solution,  15%   

...       .36 

.25 

.00 

.0 

79 

Must  of  23°  Balling  

...  18.51 

12.17 

79 

Must  of  29?8  Balling  

...  18.54 

12.20 

11.45 

93.8 

79 

Beer  wort  of  14°  Balling  . 

...     6.36 

4.38 

2.35 

53.7 

79 

Dextrose  solution,  15%  

...  12.60 

8.20 

6.00 

72.9 

79 

Saccharose  solution,  15%  ... 

...  11.43 

7.87 

6.00 

76.2 

79 

Lactose  solution,   15%  

...       .06 

.04 

.00 

.0 

1918] 


Cruess :   Fermentation  Organisms  of  California  Grapes 


45 


^    ^0 im tm *on  z^n  &m  3$a  -y?  ^pn  ^y 


^F7D 


Fig.  13. — Fermentation  curves  of  true  wine  yeasts  from  California  grapes. 

The  effect  of  a  moderately  high  temperature  on  yeast  67  is  shown 
by  the  analyses  of  the  fermented  liquids,  from  which  it  is  seen  that 
the  absolute  amounts  of  alcohol  formed  by  this  yeast  are  small  and 
that  the  per  cents  of  the  theoretical  yields  obtained  were  very  small. 
Thus  at  33°  C  it  not  only  gives  very  incomplete  fermentations,  but 
is  also  very  wasteful  of  the  sugar  that  it  ferments.  Yeast  75  gave 
the  highest  yield  of  alcohol,  14.53  per  cent.  None  of  the  yeasts  gave  as 
much  alcohol  in  must  of  30°  Balling,  at  33°  C  as  they  did  at  24°  C. 
Contrasted  with  the  yeasts  discussed  in  the  previous  pages  the  S. 
ellipsoideus  yeasts  gave  larger  yields  of  alcohol  and  in  general  a  larger 
per  cent  of  the  sugar  destroyed  was  transformed  into  alcohol. 


46 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 


Comparison  of  Fermentations  at  33°  C  and  24°  C  (all  Varieties) 

The  nineteen  fermentation  organisms  discussed  in  the  preceding 
pages  were  transferred  to  grape  must  of  29.8°  Balling,  and  the  pro- 
ducts of  fermentation  at  24°  C  and  33°  C  studied  and  compared.  The 
results  appear  in  table  11. 

TABLE  11 

Comparison  of  Products  of  Fermentation  of  California  Yeasts  in  Grape 

Must  of  30°  Balling  Fermented  at  24°  C  and  33°  C 


Attenuation 
in  grams 
per  100 
Organism  c.c. 

35.  S.    apiculatus    8.31 

72.  S.    apiculatus    6.80 

65.  Mycoderma   species    ....     2.70 

68.  Mycoderma   species    ... .     4.20 

70.  Mycoderma    species    ....     3.40 

71.  Mycoderma    species    ....     8.20 

73.  Mycoderma    species    ....  23.90 

76.  Mycoderma    species    ....     3.20 

78.  Mycoderma    species    ....  10.10 
37.  Torula  species  3.70 

77.  Torula  species   6.47 

36.  S.   pastorianus   2.80 

69.  Willia   anomala   5.11 

64.  S.   ellipsoideus   24.60 

66.  S.    ellipsoideus   22.00 

67.  S.   ellipsoideus   24.01 

74.  S.   ellipsoideus   22.50 

75.  S.   ellipsoideus   22.70 

79.  S.   ellipsoideus   25.30 

35.  S.   apiculatus    6.15 

72.  S.    apiculatus    1.20 

65.  Mycoderma  vini   0.00 

68.  Mycoderma  vini   7.84 

70.  Mycoderma  vini   2.05 

71.  Mycoderma  vini   

73.  Mycoderma  vini   14.75 

76.  Mycoderma   vini   00.00 

78.  Mycoderma  vini   14.13 

37.  Torula  species   05 

77.  Torula  species  3.85 

36.  S.   pastorianus   1.65 

69.  Willia  Anomala  2.92 

64.  S.  ellipsoideus  21.40 

00.  S.    ellipsoideus    19.75 

07.  S.  ellipsoideus  3.85 

74.  S.   ellipsoideus  20.95 

7;1.  S.    Hlipsoi<l<Mis    22.10 

79.  S.    cllipso'i-lcus    18.54 


Theoretical 
yield  of 
alcohol, 
volume, 
per  cent 

5.46 

Alcohol 
obtained, 
volume, 
per  cent 

2.60 

Per  cent  of 
theoretical 

yield 
obtained 

47.6 

Temperature 
24°  C 

4.47 

3.20 

72.7 

24°  C 

1.70 

0.00 

00.0 

24°  C 

2.70 

1.20 

44.4 

24°  C 

2.20 

.60 

27.3 

24°  C 

5.40 

3.20 

59.2 

24°  C 

15.70 

13.50 

86.0 

24°  C 

2.10 

.65 

30.9 

24°  C 

6.64 

4.05 

60.9 

24°  C 

2.43 

.30 

12.3 

24°  C 

4.25 

1.05 

24.7 

24°  C 

1.85 

0.00 

00.0 

24°  C 

3.35 

1.05 

31.3 

24°  C 

16.20 

15.60 

96.3 

24°  C 

14.40 

14.10 

97.9 

24°  C 

15.80 

15.30 

96.8 

24°  C 

14.80 

14.20 

95.2 

24°  C 

14.90 

14.40 

96.6 

24°  C 

16.60 

15.55 

93.7 

24°  C 

.79 

.30 

37.9 

33°  C 

4.03 

2.35 

58.30 

33°  C 

0.00 

0.00 

00.0 

33°  C 

5.15 

4.50 

89.30 

33°  C 

1.35 

1.00 

74.50 

33°  C 

7.90 

33°  C 

9.70 

6.80 

70.10 

33°  C 

0.00 

0.00 

00.00 

33°  C 

9.29 

9.00 

97.90 

33°  C 

.03 

0.00 

00.00 

33°  C 

2.53 

.65 

25.70 

33°  C 

1.08 

.00 

00.00 

33°  C 

1.90 

1.00 

52.60 

33°  C 

13.90 

12.80 

92.10 

33°  C 

12.90 

11.40 

88.40 

33°  C 

2.53 

1.30 

51.30 

33°  C 

13.70 

11.40 

83.20 

33°  C 

14.53 

33°  C 

12.20 

11.45 

93.80 

33°  C 

1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  47 

TABLE   11— (Continued) 

Loss  in  fixed 
acid  during 
Volatile  acid, grams         fermentation, 
Organism  per  100  c.c.  grams  per  100  c.c.  Temperature 

35.  S.  apiculatus    110  —.10  24°  C 

77.  S.  apiculatus    100  .05  24°  C 

65.  Mycoderma   vini   020  .06  24°  C 

68.  Mycoderma   vini   050  —.19  24°  C 

70.  Mycodrema  vini   030  — .10  24°  C 

71.  Mycoderma  vini  043  — .07  24°  C 

73.  Mycoderma  vini   097  .02  24°  C 

76.  Mycoderma  vini   020  .04  24°  C 

78.  Mycoderma  vini   '    .056  — .08  24°  C 

37.  Torula  species   084  .06  24°  C 

77.  Torula  species  020  — .02  24°  C 

36.  S.   pastorianus   030  —.04  24°  C 

69.  Willia  anomala  080  —.06  24°  C 

64.  S.   ellipsoideus   060  .06  24°  C 

66.  S.   ellipsoideus   050  .10  24°  C 

67.  S.   ellipsoideus   080  .12  24°  C 

74.  S.   ellipsoideus   078  .11  24°  C 

75.  8.    ellipsoideus   070  .06  24°  C 

79.  S.   ellipsoideus   090  .02  24°  C 

Discussion  of  Results  in  Table  11. — Organisms  35,  72,  73,  76,  37, 
64,  66,  67,  and  74  gave  higher  percentages  of  the  theoretical  yield  at 
24°  C  than  at  33°  C.  Organisms  68,  70,  77,  and  69  gave  higher  per- 
centages of  the  theoretical  at  33°  C  than  at  24°  C  and  organism  79 
gave  approximately  the  same  percentages  at  33°  C  and  24°  C,  while 
organisms  65  and  36  gave  no  alcohol  at  either  temperature. 

In  regard  to  the  absolute  amounts  of  alcohol  formed,  the  relations 
are  somewhat  different.  Yeasts  68,  70,  71,  and  78  gave  higher  yields 
of  alcohol  at  33°  C  than  at  24°  C.  All  of  these  are  Mycoderma  forms. 
This  might  indicate  that  these  forms  have  a  higher  optimum  than  the 
other  forms  tested.  All  other  forms  tested  gave  higher  yields  of 
alcohol  at  24°  C  than  at  33°  C.  The  results  are  especially  striking 
with  8.  ellipsoideus,  number  67,  in  this  respect.  It  gave  1.3%  alcohol 
at  33°  C  and  15.3%  at  24°  C. 

The  wild  yeasts,  i.e.,  the  8.  apiculatus,  Mycoderma,  Torula,  S.  pas- 
torianus, and  Willia  yeasts,  with  the  exception  of  organism  73,  all 
gave  small  amounts  of  alcohol  and  were  very  wasteful  of  the  sugar 
fermented.  The  percentage  of  the  theoretical  yield  varied  from  0% 
to  72%.  On  the  other  hand,  the  8.  ellipsoideus  yeasts  gave  fairly 
high  amounts  of  alcohol,  varying  from  14.1%  to  15.6%  at  24°  C,  and 
all  gave  over  93%  of  the  theoretical  yield  of  alcohol  from  the  sugar 
destroyed  at  24°  C.     These  facts  emphasize  the  necessity  of  favoring 


48  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

the  growth  of  the  true  wine  yeasts  and  discouraging  the  development 
of  the  wild  yeasts. 

It  is  commonly  held  by  enologists  in  general  that  wild  yeasts  as  a 
rule  destroy  large  amounts  of  tartaric  acid  and  form  high  amounts  of 
volatile  acid  during  fermentation.  To  test  this  belief  with  the  Cali- 
fornia wild  yeasts  under  observation,  analyses  of  the  original  unfer- 
mented  must  used  in  the  above  tests  and  of  the  fermented  liquids  were 
made.  Contrary  to  expectations  they  did  not  destroy  abnormally  large 
amounts  of  fixed  acid  during  fermentation,  so  that  it  is  possible  that 
the  low  yield  of  fixed  acid  in  naturally  fermented  wines  compared  to 
those  fermented  with  pure  yeast  and  sulfur ous  acid  may  be  due  to  other 
causes  than  the  development  of  the  wild  yeasts.  For  example,  it  may 
be  due  to  bacteria,  or  the  higher  yield  of  fixed  acid  in  wines  in  which 
sulfurous  acid  has  been  used  may  be  due  to  the  chemical  effect  of  the 
sulfurous  acid  on  the  cream  of  tartar  which  it  may  protect  from  preci- 
pitation by  the  alcohol  of  the  wine.  The  fact  that  the  use  of  sulfurous 
acid  in  wine  making  gives  a  higher  yield  of  fixed  acid  in  the  finished 
wine  made  from  the  same  grapes  by  natural  fermentation,  and  the 
results  of  the  above  tests,  simply  cast  doubt  on  the  theory  held  that 
the  low  yield  of  fixed  acid  in  naturally  fermented  wines  is  due  to 
the  activity  of  the  wild  yeasts.  They  do  not  necessarily  prove  the 
contrary  view,  because  of  the  small  number  of  yeasts  under  obser- 
vation. 

The  largest  amount  of  volatile  acid  was  formed  by  the  S.  apicu- 
latus  yeast,  which  gave  0.11%.  This,  in  itself,  is  considerably  below 
the  legal  limit  of  0.140%  for  red  wines  and  0.120%  for  white  wines. 
The  real  point  to  note,  however,  is  that  the  apiculatus  yeast  develops 
in  natural  wine  fermentations  before  the  true  wine  yeast  has  had  a 
chance  to  carry  on  any  great  amount  of  fermentation,  so  that  the 
volatile  acid  formed  by  the  apiculatus  yeast  is  added  to  that  later 
formed  by  the  true  wine  yeast  and  other  yeasts.  In  this  way  the 
volatile  acid  may  rise  dangerously  high.  Added  to  this,  is  the  fact 
that  the  apiculatus  yeast  produces  compounds  during  fermentation 
that  are  prejudicial  to  the  activity  of  the  true  wine  yeast  and  so  cause 
it  to  behave  abnormally  and  produce  larger  amounts  of  volatile  acid 
than  it  otherwise  would.  It  may  be  stated  that  wine  ceases  to  be  com- 
mercially salable  when  it  contains  more  than  0.14%  volatile  acid. 

From  the  data  and  discussions  given  in  the  foregoing  pages  on  the 
properties  of  California  yeasts,  it  may  be  said  that  the  yeasts  of  the 
8.  (Uipsmdeus  or  true  wine  yeast  type  are  the  only  ones  so  far  found 


1918] 


Cruess:   Fermentation  Organisms  of  California  Grapes 


49 


4C)            9 

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Fig.  14. — Fermentation  curves  of  true  wine  yeasts  from  California  grapes. 

that  are  suitable  for  the  production  of  wine.  Although  several  of  the 
wild  yeasts  produce  fairly  large  amounts  of  alcohol  and  low  volatile 
acid  during  fermentation,  they  can  not  be  used  in  wine  making,  be- 
cause they  produce  undesirable  flavors  and  odors.  An  objection  to  the 
California  8.  ellipsoideus  yeasts  is  the  fine  grained  character  of  their 
sediments,  giving  slow  clearing  after  fermentation  and  difficulty  in 
racking.  Another  defect  is  that  some  of  them  seem  to  be  weakened 
very  perceptibily  by  such  a  moderate  temperature  as  90°  F  (32°  C). 


50 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


2Q 12Q. IW. &na &3Q. ZiW     TifiQ 7>W    dQQ    "HP 


366)     1UO   44b 


Fig.  15. — Fermentation  curves  of  true  wine  yeasts  from  California  grapes. 

Several  varieties  of  8.  ellipsoideus  isolated  in  France  and  cultures 
of  which  are  in  the  University  collection  give  much  better  results  than 
any  of  the  yeasts  isolated  from  California  grapes  in  this  laboratory. 
Consequently  i1  seems  desirable  to  use  one  of  the  French  varieties 
rather  than  any  of  the  above  California  yeasts  for  commercial  fer- 
mentation of  grapes  for  wine. 


1918]  Cruess :   Fermentation  Organisms  of  California  Grapes  51 

IV.     INFLUENCE  OF  LOCALITY  ON  THE  CHARACTER  OF 

THE  MICRO-ORGANSIMS  ON  GRAPES 

Methods  of  Taking  Samples6 

Samples  of  grapes  were  obtained  from  Davis,  Fresno,  and  El 
Centro,  all  localities  in  California.  Two  Mason  fruit  jars  of  one  quart 
capacity  each  were  plugged  with  cotton  wool,  packed  into  a  small  box 
with  excelsior,  and  the  box  and  contents  were  sterilized  at  150°  C  in 
a  dry  heat  sterilizer.  Two  such  jars  were  sent  to  each  of  the  above 
localities.  Several  small  bunches  of  grapes  were  cut  and  allowed  to 
fall  into  the  open  jars.  The  cotton  plugs  were  then  replaced.  The 
grapes  were  sent  to  Berkeley  where  the  numbers  of  the  different 
groups  of  yeasts  present  were  determined.  Samples  from  Contra 
Costa  County,  California,  were  taken  in  sterile  paper  bags.  The  organ- 
isms on  grapes  from  Ripon,  San  Joaquin  County,  California,  were 
determined  at  the  vineyard. 

Method  of  Counting  Living  Cells  on  Grapes 
The  same  sort  of  grape  must  agar,  and  sterile  petri  dishes  pre- 
viously described  were  employed  in  the  counting  tests.  In  addition 
to  the  above  apparatus  a  number  of  sterile  one  c.c.  pipettes,  and  test 
tubes,  each  containing  9  c.c.  of  sterile  water  were  necessary.  On 
arrival  at  the  laboratory,  the  grapes  were  crushed  in  the  jars  by  means 
of  a  sterile  pestle.  One  cubic  centimeter  of  the  fresh  juice  was  re- 
moved by  means  of  a  sterile  1  c.c.  pipette  and  transferred  to  a  tube 
of  9  c.c.  sterile  water.  With  another  sterile  1  c.c.  pipette  one  cubic 
centimeter  of  the  liquid  in  the  first  tube  of  water  was  transferred  to 
a  second  tube.  This  process  was  carried  progressively  to  a  third, 
fourth,  and  occasionally  a  fifth  tube,  depending  on  the  condition  of  the 
grapes.  This  gave  a  dilution  of  1 :10  in  the  first  tube,  1 :100  in  the 
second  tube,  1  :1000  in  the  third,  and  1 :10,000  in  the  fourth.  From 
each  of  these  tubes  one  cubic  centimeter  of  the  liquid  was  removed  to 
separate  petri  dishes.  Melted  agar  must  at  40°  C  to  45°  C  was  poured 
into  each  plate  and  the  plates  set  away  to  allow  development  of  the 
individual  cells  into  colonies.  Counts  of  the  number  of  colonies  and 
notes  of  the  numbers  of  each  type  of  organism  present  were  made. 
From  the  dilutions  used  it  was  possible  to  calculate  the  number  of 
active  cells  of  each  kind  of  micro-organism  present  in  a  unit  volume 
of  the  original  liquid  (see  plate  1). 


6  The  writer  wishes  to   express  his  appreciation   of  the  aid  given  him  by 
Messrs.  Flossfeder,  Way,  and  Packard  in  taking  samples. 


52 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


Results  op  Counting 
The  results  of  the  countings  made,  appear  in  the  following  tables 


TABLE  12 

Micro-Organisims  on  Grapes  from  Davis 

Sample  number  1,  Sample  number  2, 

first  crop,  second  crop, 

Muscat  grapes,  Muscat  grapes, 

Organism                                                cells  per  c.c.  cells  per  c.c. 

Penicillium,  green  mold  (probably  glaucum                  none  none 

Aspergillus  mold  (probably  niger)  none  1,000 

Mucor  mold  20,000  none 

Dematium  mold  130,000  105,000 

Powdery  white  mold  none  3,000 

S.  apiculatus   (a  wild  yeast)   less. than  100*  less  than  100 

Other  wild  yeasts  200,000  

True  wine  yeast  (S.  ellipsoideus)  none  none 

*  The  dilutions  below  1:100  were  so  badly  overgrown  with  the  dematium  mold  that  it 
was  not  possible  to  differentiate  the  yeasts  present,  so  that  "less  than  100  per  c.c."  may 
mean  from  none  to  100  per  c.c.  It  signifies  that  no  S.  apiculatus  or  true  wine  yeasts  appeared 
on  the  dilution  of  1:100.  There  were  no  true  wine  yeast  cells  present,  however,  because 
all  of  the  samples  were  plated  out  again  during  fermentation  and  in  this  way  true  wine  yeast 
cells  were  proven  to  be  absent.  Added  proof  of  the  absence  of  true  wine  yeast  is  given 
by  the  fact  that  the  crushed  grapes  did  not  undergo  true  wine  yeast  fermentation  but  after 
a   feeble  wild  yeast   fermentation  became   moldy   and  rotted. 

TABLE  13 
Micro-Organisms  on  Grapes  from  El  Centro 

Sample  number  1,  Sample  number  2, 

Organism  cells  per  c.c.  cells  per  c.c. 

Penicillium   (olive   green   mold)    2,400,000  20,000,000 

Penicillium   (blue  green  mold)  none  50,000 

Fusarium-like  mold  30,000  none 

Aspergillus  mold  40,000  100,000 

S.  apiculatus  less  than  100  less  than  100 

Wild  yeasts,  Mycoderma  vini  type  ....  400,000  8,000,000 

True  wine  yeasts  (S.  ellipsoideus)   none  none 

There  were  no  true  wine  yeasts  present  because  the  grapes  did 
not  undergo  a  wine  yeast  fermentation  but  simply  became  very  moldy 
and  finally  rotted. 

TABLE  14 
Micro-Organisms  from  Grapes  Grown  at  Fresno 

White  grapes,  Red  grapes, 

Organism                                              cells  per  c.c.  cells  per  c.c. 

Penicillium    (olive  green   mold)    10,000  15,000 

Aspergillus  mold  : 10,000  1,000 

Fusarium-like  mold  10,000  4,000 

Dematium   mold  7,500  560,000 

Mucor    mold    200  less  than  100 

S.    apiculatus    less  than  100  less  than  100 

True  wine  yeast    (S.   ellipsoideus  less  than  100  less  than  100 

These  grapes  underwent  true  wine  yeast  fermentation,  proving 
the  presence  of  a  few  8.  ellipsoideus. 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  53 

TABLE  15 
Micro-Organisms  on  Grapes  from  Vineyard  of  J.  Swett  &  Son,  Martinez 

Alicante  Sauvignon  Isabella 

Bouschet  grapes,  verte  grapes,  grapes, 

Organism  cells  per  c.c.       cells  per  c.c.        cells  per  c.c. 

Penicillium    (green   mold)    5,000  3,000  15 

Mucor   (gray  mold)    2,000  none  none 

Dematium   (tree  mold)   17,000  22,000  18,000 

Aspergillus  mold  none  none  200 

Monilia    mold    12,000  30,000  10,000 

Wild  yeasts  14,000  36,000  100 

True  wine  yeast   (S.  ellipsoideus  a  few  a  few  a  few 

These  grapes  underwent  normal  alcoholic  fermentation,  proving 
the  presence  of  S.  ellipsoideus. 

TABLE  16 
Micro-Organisms  from  Grapes  Grown  at  Kipon* 

Organism  Cells  per  c.c. 

Penicillium   (blue  green  mold)    1,700 

Mucor   mold    100 

Aspergillus  mold   less  than  100 

Wild   yeasts    2,600 

True  wine  yeasts  less  than  100  but  more  than  0 

*  The  above  counts  were  made  by  plating  the  grapes  in  the  vineyard. 

Yeasts  From  the  Tulare  Experiment  Station 

In  1906  grapes  were  gathered  at  the  Tulare  substation  by  Pro- 
fessor Bioletti7  under  conditions  that  precluded  contamination  by 
organisms  not  present  on  the  grapes.  The  yeasts  present  on  them 
were  isolated  by  H.  C.  Holm  and  their  properties  studied.  All  of 
them  were  found  to  be  wild  yeasts  of  very  low  fermenting  power  and 
all  gave  undrinkable  fermented  musts.    No  true  wine  yeast  was  found. 


Discussion  of  Tables  12  to  16,  Inclusive 

The  grapes  from  Davis,  Fresno,  El  Centro,  Ripon,  and  Martinez 
were  all  allowed  to  ferment  after  crushing  and  plating  tests  were  made 
during  fermentation  in  order  to  obtain  the  true  wine  yeast  if  present. 
The  Davis  grapes  and  the  El  Centro  samples  did  not  undergo  true 
yeast  fermentations,  but  after  a  short  wild-yeast  fermentation,  became 
very  moldy  and  finally  rotted.  The  grapes  from  the  other  localities 
underwent  normal  true  wine  yeast  fermentations  and  the  yeasts  of 
this  type  present  were  found  on  dilutions  in  agar  must  made  during 
fermentation. 


7  Bioletti,  F.  T.,  and  Holm,  H.  C,  Calif.  Exp.  Sta.  Bull.  197,  pp.  169, 175, 1908. 


54  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

Wine  is  not  made  in  the  regions  in  the  immediate  vicinity  of  Tulare 
and  El  Centro,  and  none  is  made  at  the  University  Farm,  Davis.  The 
grapes  from  Fresno,  Ripon,  and  Martinez  came  from  places  near 
wineries  and  on  all  of  these  samples  true  wine  yeasts  were  found. 
These  tests  indicate  that  grapes  from  regions  remote  from  wineries 
have  smaller  numbers  of  true  wine  yeast  cells  on  their  surfaces  than 
grapes  from  wine  making  regions.  Reasoning  from  these  facts  it  is 
probable  that  the  grapes  gathered  during  the  beginning  of  the  season 
will  have  fewer  true  wine  yeast  cells  than  those  picked  later  in  the 
crushing  season.  Thus  the  use  of  pure  yeast  is  more  necessary  on 
grapes  from  regions  distant  from  wineries  and  on  grapes  gathered 
during  the  forepart  of  the  season  that  it  is  on  those  picked  in  wine 
making  regions  after  the  season  has  progressed  for  several  weeks. 
The  figures  given  in  Part  IV  indicate  the  influence  of  the  time  of 
picking  on  the  numbers  of  yeasts,  etc.,  present. 

All  the  samples  tested  above  gave  a  great  preponderance  of  molds 
and  wild  yeasts  over  the  true  wine  yeasts.  It  can  easily  be  seen  that 
if  there  are  several  hundred  thousand  wild  yeasts  and  a  million  or 
two  mold  cells  for  each  one  to  a  hundred  true  yeast  cells,  as  was 
actually  the  case  in  several  instances,  the  true  wine  yeasts  will  have 
overwhelming  odds  against  which  to  develop.  Where  such  a  condi- 
tion of  affairs  exists  the  wild  yeasts  must  of  necessity  cause  consider- 
able damage.  It  is  not  probable  that  the  molds  do  a  great  deal  of 
damage  after  crushing  because  the  conditions  are  not  very  favorable 
for  their  growth  in  the  crushed  grapes,  but  they  undoubtedly  de- 
preciate the  value  of  grapes  for  wine  making  during  long  shipments. 

In  summarizing  the  above  data  it  may  be  stated  that  grapes,  both 
from  wine  making  regions  and  from  those  in  which  wine  is  not  made, 
are  deficient  in  true  wine  yeasts  and  carry  a  large  number  of  molds 
and  wild  yeasts  prejudicial  to  the  making  of  the  best  wines.  Grapes 
from  regions  remote  from  wineries  seem  to  bear  smaller  numbers  of 
the  true  wine  yeasts  than  those  from  wine  making  localities. 

V.     INFLUENCE   OF   THE   STAGE   OF   RIPENESS   ON   THE 
CHARACTER  OF  THE  MICRO-ORGANISMS  ON  GRAPES 

Sampling 

Grapes  were  gathered  in  sterile  paper  bags  from  a  Zinfandel  vine 
in  a  large  vineyard  at  Muir  Station  in  Contra  Costa  County,  Cali- 
fornia, in  1912.  One  sample  was  taken  when  the  grapes  were  green, 
a  second  when  they  had  begun  to  turn,  and  the  third  in  the  middle  of 


1918] 


Cruess :    Fermentation  Organisms  of  California  Grapes 


55 


September  when  the  grapes  were  ripe.  Since  the  samples  were  all 
from  the  same  vine,  they  may  be  taken  as  being  more  or  less  com- 
parative. A  sample  was  taken  from  an  Alicante  Bouschet  vine  in 
the  same  vineyard  as  the  Zinfandel  when  the  grapes  were  green  and 
a  second  sample  in  the  middle  of  September  from  another  vine  of  the 
same  variety  in  the  vicinity  of  the  first  one. 

The  grapes  were  crushed  in  the  laboratory,  using  precautions 
against  outside  infection,  and  counts  of  the  active  cells  present  were 
made  as  in  the  tests  recorded  in  Part  III. 

Eesults 
The  results  of  the  tests  appear  in  the  accompanying  tables . 

TABLE  17 
Numbers  of  Micro-Organisms  on  Grapes  at  Different  Stages  of  Ripeness 


Ripeness  of 
grapes  at 
sampling 

Type  of 

organisms 

found 

Cells  per  c.c. 

of  must, 

Zinfandel  grapes 

Cells  per  c.c. 
of  must,  Alicante 
Bouschet  grapes 

r 

Molds 

1,040,000 

100 

Hard,  greenJ 

"Wild  yeasts 

less  than  10 

less  than  10 

I 

S.  ellipsoideus 

none 

none 

r 

Molds 

1,000,000 

ginning  to  eolorJ 

Wild  yeasts 

175,000 

1 

S.  ellipsoideus 

none 

f 

Molds 

190 

22,000 

RipeJ 

Wild  yeasts 

3,360 

26,000 

1 

S.  ellipsoideus 

less  than  ls 
but  present 

a  few 

The  crushed  samples  were  allowed  to  stand  in  sterile  jars  plugged 
with  cotton.  Samples  taken  when  the  grapes  were  hard  green  and 
beginning  to  color,  molded  and  putrefied,  but  did  not  ferment,  indi- 
cating absence  of  8.  ellipsoideus.  The  samples  of  ripe  grapes  both 
fermented,  showing  presence  of  8.  ellipsoideus. 

On  the  green  grapes  molds  were  about  the  only  kind  of  organisms 
present.  When  the  grapes  began  to  turn  in  color  the  wild  yeasts  had 
made  their  appearance  in  large  numbers,  but  no  true  yeasts  were  in 
evidence.  On  the  ripe  grapes  were  found  chiefly  wild  yeasts  and 
molds  and  in  addition  to  these  a  few  8.  ellipsoideus  cells,  not  enough 
to  develop  on  the  plates  of  dilution  1 :10,  but  still  enough  to  cause 
the  grapes  to  undergo  a  true  yeast  fermentation  after  standing  several 
days  in  a  sterile  flask  plugged  with  cotton  wool. 


56  University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 

The  surfaces  of  the  hard  green  grapes  are  very  poor  places  for  the 
development  of  micro-organisms;  and  molds  predominate  probably 
because  they  are  merely  present  in  the  dust  on  the  surface  of  the 
grapes.  As  the  grapes  soften  during  ripening  some  of  them  are  broken 
by  birds  and  insects  and  the  yeasts,  etc.,  develop  in  the  broken  berries. 
From  these  the  cells  are  carried  to  the  surfaces  of  other  grapes  by 
insects,  etc.,  so  that  as  the  season  advances  the  numbers  of  yeasts 
increase.  Even  on  the  ripe  grapes,  however,  the  numbers  of  the  true 
wine  yeast  cells,  8.  ellipsoideus,  were  small  and  were  greatly  exceeded 
by  those  of  the  molds  and  wild  yeasts.  The  data  of  this  table  do  not 
confirm  the  statement  often  made  that  the  numbers  of  micro-organisms 
increase  during  ripening. 

VI.  CHANGES  IN  THE  NUMBERS  AND  CHARACTER  OF  THE 

MICRO-ORGANISMS  ON  GRAPES  DURING  SHIPMENT 

FROM  VINEYARD  TO  CELLAR 

Burger  grapes  were  picked  at  the  vineyard  of  J.  Swett  &  Son, 
Ripon,  California,  and  dilution  plates  on  agar  were  made  at  the  vine- 
yard on  the  freshly  picked  grapes.  Counts  were  also  made  on  grapes 
from  the  same  block  of  the  vineyard  after  they  had  stood  about  eigh- 
teen hours  in  boxes.  The  sample  in  this  latter  case  was  taken  from  a 
lot  of  two  boxes  that  were  crushed  into  a  sterile  barrel.  After  the 
grapes  arrived  at  the  winery  in  Martinez  three  days  later  an  average 
sample  was  taken  and  counts  of  the  micro-organisms  present  again 
made. 

Alicante  Bouschet  grapes  were  plated  at  the  vineyard  and  on 
arrival  three  days  later  at  the  winery.  The  results  of  the  above  tests 
appear  in  tables  18  and  19. 

TABLE  18 

Change  in  Micro-Organisms  on  Burger  Grapes  After  Picking 

Grapes  after  Grapes  after 
Grapes  in                     18  hours  3  days  on 

vineyard,  in  boxes,  board  cars, 

cells  per  c.c.  cells  per  c.c.  cells  per  c.c. 

Organism  of  must  of  must  of  must 

1.  Penicillium   mold   1,700  145,000  ] 

2.  Mucor  mold  100  50,000  ( 

3.  Aspergillus  mold  none  none  ^9,200,000 

4.  Dematium  mold  less  than  1  72,000 

5.  Myeodera    forms    and    Torula 

yeasts   2,500  160,000  4,700,000 

0.  S.  apiculatus  yeast  less  than  1  less  than  10  less  than  1,000 

7.  True  wine  yeast  (S.  ellipsoideus)   less  than  1  less  than  100  500,000 

8.  Vinegar   bacteria    none  none  800,000 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  57 

TABLE  19 
Change  in  Micro-Organisms  on  Alicante  Bouschet  Grapes  During  Shipment 

Grapes  three  days 
Grrapes  in  vineyard,  later  at  cellar, 

cells  per  c.c.  cells  per  c.c. 

Organism  of  must  of  must 

1.  Penicillium  mold  320,000  2,500,000 

2.  Mycoderma  forms  125  5,400,000 

3.  Other  wild  yeasts  none  less  than  1,000 

4.  True  wine  yeast  (S.  ellipsoideus) 25  440,000 

5.  Vinegar   bacteria    none  less  than  100 

The  effect  of  standing  over  night  in  the  boxes  was  to  greatly 
increase  the  numbers  of  wild  yeasts  and  molds  present  without  mate- 
rially affecting  the  quantity  of  true  wine  yeast  present.  No  doubt  a 
great  deal  of  the  mold  and  wild  yeast  cells  came  from  the  sides  and 
bottoms  of  the  boxes,  where  they  had  collected  from  broken  and  moldy 
berries  during  previous  shipments.  Thus,  it  may  be  seen  how  grapes 
almost  free  from  micro-organisms  could  acquire  large  numbers  by 
standing  in  boxes  a  few  hours.  Some  of  the  increase  is,  of  course, 
due  to  normal  increase  of  the  cells  present  on  the  surface  of  the  grapes 
and  to  increase  of  organisms  in  the  grapes  broken  during  picking. 

The  grapes  were  three  days  on  the  road  from  the  vineyard  to  the 
winery  and  arrived  in  a  moldy  condition,  but  in  no  worse  state  than 
other  Burger  grapes  shipped  under  like  conditions.  The  plating  tests 
bore  out  what  the  appearance  indicated,  namely,  the  presence  of  great 
numbers  of  molds,  9,000,000  per  cubic  centimeter;  wild  yeasts, 
4,700,000,  and  even  large  numbers  of  vinegar  bacteria,  800,000  per 
cubic  centimeter.  The  wine  yeast  was  present  in  less  numbers  than 
any  of  the  other  organisms,  there  being  only  500,000  of  them  per 
cubic  centimeter. 

The  Alicante  Bouschet  grapes  gave  similar  results,  but  being  firmer 
grapes  they  arrived  in  better  condition  than  the  Burgers,  as  was 
shown  by  their  appearance  and  by  counting  tests. 

A  method  of  controlling  the  micro-organisms  on  grapes  during 
shipment  is  discussed  under  Part  VII. 


VII.     CHARACTER  AND  NUMBERS  OF  MICRO-ORGANISMS 

ON  GRAPES  AS  RECEIVED  AT  THE  WINERY  DURING 

THE  SEASONS  OF  1911  AND  1912 

Samples  of  the  freshly  crushed  grapes  from  two  wineries  in  Contra 
Costa  County  were  taken  in  1911  and  1912  and  counts  of  the  active 
cells  present  were  made  with  the  results  given  in  the  following  table : 


58 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


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1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  59 

These  tests  justify  the  statement  that  grapes  ordinarily  arrive  at 
the  winery  with  a  preponderance  of  the  undesirable  organisms  as  com- 
pared with  the  desirable  true  wine  yeast,  8.  ellipsoideus.  The  actual 
numbers  of  all  molds,  wild  yeasts,  etc.,  are  less  on  grapes  gathered 
near  the  winery  than  on  those  shipped  considerable  distances  before 
crushing;  but  the  ratio  of  molds  and  wild  yeasts  to  true  yeasts  is 
greater  on  clean  grapes  than  on  moldy  ones  judging  from  the  above 
tests.  Therefore,  there  is  just  as  much,  or  even  a  greater  need  for 
the  use  of  pure  yeast  with  clean  grapes  than  with  grapes  in  poor 
condition,  although  it  is  extremely  desirable  to  check  the  growth  of  the 
undesirable  and  promote  the  development  of  the  desirable  types  in 
both  cases. 


VIII.     EXPERIMENTS  UPON  THE  CONTROL  OF  MICRO- 
ORGANISMS ON  GRAPES  FOR  WINE  MAKING 

Before  Shipment 

Burger  and  Alicante  Bouschet  grapes  from  the  vineyard  of  J. 
Swett  &  Son  were  used  in  the  tests  discussed  below. 

Several  boxes  of  Burgers  were  crushed  into  a  barrel  that  had  been 
sterilized  with  strong  potassium  metabisulfite  solution  and  rinsed  out 
with  clean  water  to  remove  the  metabisulfite.  To  the  crushed  grapes 
was  added  potassium  metabisulfite  at  the  rate  of  12  ounces8  per  ton 
of  grapes.  This  was  added  in  the  form  of  a  10  per  cent  solution.  Four 
clean  Mason  fruit  jars  were  filled  with  the  crushed  grapes  and  sul- 
fited  at  the  rates  of  6,  8,  9,  and  14  ounces  per  ton.  The  metabisulfite 
is  decomposed  by  the  tartaric  acid  of  the  grapes  to  give  sulfur  dioxide 
and  cream  of  tartar. 

Several  bunches  of  Alicante  Bouschet  grapes  were  crushed  into  a 
Mason  jar  and  were  treated  at  the  rate  of  about  eight  ounces  of  meta- 
bisulfite per  ton.  The  treated  Burgers  and  Alicante  Bouschet  were 
shipped  from  Ripon  to  Martinez  in  the  same  car  with  untreated 
grapes.  Counts  of  the  micro-organisms  on  the  grapes  before  treatment 
and  on  the  treated  and  untreated  grapes  at  the  winery  upon  their 
arrival  three  days  later  were  made. 


s  Twelve    ounces    of    K2  S2  03    (potassium   metabisulfite)    per   ton    corresponds 
to  approximately  190  milligrams   S02    (sulfur  dioxide)    per  kilogram   of   grapes. 


60 


University  of  California  Publications  in  Agricultural  Sciences        [Vol.  4 


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1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  61 

The  Burger  grapes  in  the  jars  all  arrived  in  good  condition,  i.e., 
with  no  visible  mold  growth  and  no  fermentation ;  in  fact,  none  of  the 
treated  grapes  were  moldy  or  fermenting.  The  untreated  grapes  of 
both  varieties  shipped  in  the  ordinary  way  were  visibly  moldy  and 
in  some  bunches  the  odors  of  fermentation  and  acetic  acid  were 
noticeable. 

The  amounts  of  sulfurous  acid  used  were  small  and  yery  much 
below  the  legal  amount  of  350  milligrams  per  liter  allowed  in  finished 
wine  in  France.  Analyses  of  the  samples  for  sulfur  dioxide  on  arrival 
at  the  cellar  are  tabulated  in  table  24.  The  grapes  in  the  barrel  were 
sulfited  twelve  ounces  per  ton,  which  is  the  equivalent  to  190  milli- 
grams of  sulfurous  acid  per  kilogram.  On  arrival  at  the  cellar  they 
contained  only  110  milligrams  of  total  sulfurous  acid  per  liter.  In 
other  words  almost  one-half  of  the  sulfurous  acid  had  disappeared 
during  shipment. 

TABLE  22 
Sulfurous  Acid  in  Treated  Grapes  on  Arrival  at  the  Cellar 

Total  sulfurous        Free  sulfurous 
acid  in  milligrams  acid  in  milligrams 
Sample  per  liter  per  liter 

1.  Barrel  of  sulfited  Burgers  110  45 

2.  Jar   number   1,   Burger 48  16 

3.  Jar  number  2,  Burger  60  20 

4.  Jar  number  3,  Burger  68  •  32 

5.  Jar  number  4,  Burger  160  64 

It  was  thought  possible  that  the  sprinkling  of  a  little  dilute  potas- 
sium metabisulfite  solution  over  the  grapes  in  the  boxes  before  ship- 
ment might  protect  them  against  mold  growth,  etc.  Accordingly, 
about  150  c.c.  of  a  5  per  cent  solution  of  potassium  metabisulfite  was 
sprinkled  throughout  the  bottom,  center,  and  top  of  each  of  several 
boxes  of  Burger  grapes  and  these  were  shipped  with  the  untreated 
grapes.  Contrary  to  expectations,  they  arrived  in  about  as  poor  con- 
dition as  the  untreated  grapes.  The  cause  for  the  ineffectiveness  of 
the  treatment  probably  was  that  the  metabisulfite  solution  dried  out 
rapidly  during  shipment  and  thus  became  of  little  use  in  checking 
mold  growth. 

Discussion  of  Tables  19,  20,  and  21. — From  the  results  of  these 
tables  it  can  be  seen  that  the  wild  yeasts  and  molds  increase  enor- 
mously during  shipment  under  the  conditions  of  the  above  experi- 
ments, which  represent  a  fair  average  of  the  present  commercial 
methods  of  shipping.  A  moderate  amount  of  sulfurous  acid  held  the 
undesirable  organisms  in  check  so  well  that  none  of  these  could  be 
found  in  one  of  the  samples  of  treated  grapes  and  only  a  few  in  the 


62  University  of  California  Publications  in  Agricultural  Sciences        [Vol.4 

other.    The  true  wine  yeasts  were  not  entirely  suppressed  but  actually 
increased  in  numbers,  though  fermentation  had  not  started. 

This  method  could  be  applied  commercially  by  crushing  the  grapes 
into  wooden  or  iron  tank  cars  at  the  vineyard  and  at  the  same  time 
adding  sulfur ous  acid  in  the  form  of  6%  sulfurous  acid  solution  or 
potassium  metabisulfite.  For  normal  conditions  and  where  it  is  desired 
to  prevent  all  fermentation  and  growth  of  mold,  etc.,  during  shipping, 
it  would  be  advisable  to  use  from  twelve  to  sixteen  ounces  of  the 
metabisulfite  or  three-quarters  to  one  gallon  of  a  6%  sulfurous  acid 
per  ton  of  crushed  grapes.  If  it  should  be  desired  to  prevent  only  the 
growth  of  molds  and  wild  yeasts,  twelve  ounces  of  the  metabisulfite 
or  three-quarters  of  a  gallon  of  the  sulfurous  acid  6%  solution  per 
ton  would  be  sufficient  in  all  cases.  If  the  grapes  should  undergo 
wine  yeast  fermentation  no  great  harm  would  be  done.  In  fact,  one 
step  further  could  be  made  and  pure  yeast  as  well  as  metabisulfite 
could  be  added,  in  this  way  making  use  of  the  cars  as  fermenters  and 
insuring  good  fermentations.  It  is  possible  that  too  much  tannin 
would  be  extracted  in  the  shipment  of  white  grapes  by  the  crushing 
and  sulfiting  method,  but  if  no  fermentation  should  take  place  it  is  not 
likely  that  this  would  happen. 

After  Arrival  at  Cellar 

From  the  tests  recorded  in  Part  VII,  it  may  be  seen  that  the  grapes 
as  ordinarily  received  at  the  cellar  have  on  their  surfaces  large  num- 
bers of  fermentation  organisms,  most  of  which  are  of  no  value  in  wine 
making,  or  are  of  no  direct  harm  to  the  production  of  good  wine.  As 
the  season  progresses,  the  numbers  of  the  true  wine  yeast  may  increase 
in  dirty  conveyors,  in  the  boxes,  etc. ;  but  this  is  an  uncertain  method 
because  the  kind  and  the  numbers  of  the  yeasts  present  are  not  under 
control.  After  heavy  rains  such  as  in  1912,  the  numbers  of  all  yeasts 
on  the  grapes  are  diminished  by  being  washed  off.  Such  grapes  may 
undergo  all  manner  of  mold  and  wild  yeast  fermentations  because  of 
the  lack  of  true  wine  yeast.  This  was  actually  the  case  in  wineries  in 
Sonoma  County,  California,  in  1912. 

For  these  reasons,  it  becomes  advisable  to  apply  methods  of  wine 
making  that  discourage  the  growth  of  the  undesirable  types  and  favor 
the  development  of  the  true  wine  yeast.  Experiments  carried  out  in 
1911,  1913,  and  1914  and  fully  discussed  in  Bulletin  230  and  Circular 
140  of  the  California  Agricultural  Experiment  Station  demonstrated 
that  the  use  of  moderate  amounts  of  sulfurous  acid  and  the  subse- 
quent application  of  selected  pure  yeast  to  the  crushed  grapes  or 
defecated  must  gave  uniformly  good  fermentations  and  sound  wines. 


1918]  Cruess:   Fermentation  Organisms  of  California  Grapes  63 


SUMMARY  OF  PARTS  II  TO  VIII 

1.  Nineteen  different  organisms  from  California  grapes  have  been 
studied  and  their  properties  described.  The  majority  of  these  were 
found  to  be  types  that  are  harmful  in  wine  making.  The  true  wine 
yeasts  were  not  of  the  best  varieties.  If  the  yeasts  so  far  studied  are 
to  be  taken  as  representative  of  California  yeasts  in  general,  wine 
makers  should  not  rely  on  natural  fermentations  for  the  production 
of  their  wines. 

2.  Grapes  were  gathered  in  vineyards  at  Davis,  El  Centro,  Fresno, 
Ripon,  and  Martinez  under  conditions  excluding,  as  far  as  could  be, 
the  possibility  of  outside  contamination.  The  numbers  and  character 
of  the  micro-organisms  present  in  a  unit  volume  of  the  crushed  grapes 
from  each  locality  were  determined.  In  all  cases  the  undesirable 
organisms  enormously  outnumbered  the  true  wine  yeasts.  Grapes 
from  Tulare  in  1906,  Davis  in  1912,  and  El  Centro  in  1912  contained 
no  true  wine  yeasts,  indicating  that  such  yeasts  are  very  rare  in  regions 
where  wine  making  is  not  being  carried  on. 

3.  On  the  surfaces  of  the  green  grapes  examined  were  found  mostly 
molds  with  very  few  or  no  yeasts  of  any  kind.  As  the  grapes  ripened 
wild  yeasts  made  their  appearance.  The  true  wine  yeasts  were  the 
last  to  appear. 

4.  As  the  grapes  stand  in  boxes  after  picking  in  the  vineyard,  or 
on  cars  during  shipment  to  the  winery,  the  organisms  on  their  surfaces 
greatly  increase.  During  this  increase  in  numbers  of  cells  the  molds 
and  wild  yeasts  still  remain  greatly  in  excess  of  the  true  wine  yeasts. 

5.  Grapes  as  received  at  two  wineries  in  1911  and  1912  under 
normal  working  conditions  contained  large  numbers  of  molds  and  wild 
yeast  cells  and  in  one  case  vinegar  bacteria,  but  there  were  very  few 
wine  yeasts  (S.  ellipsoideus)  in  all  cases,  compared  to  the  numbers  of 
other  organisms  present. 

6.  The  molds,  wild  yeasts,  and  bacteria  may  be  eliminated  from 
the  grapes  during  shipment  by  crushing  the  grapes  at  the  vineyard 
and  adding  moderate  amounts  of  sulfurous  acid.  The  amounts  of 
sulfurous  acid  necessary  are  far  below  the  limits  allowed  by  law  in 
wine  making  countries  and  a  great  deal  of  this  disappears  during 
shipment  and  fermentation. 

7.  After  the  grapes  have  arrived  at  the  cellar,  the  development  of 
the  undesirable  types  of  fermentation  organisms  may  be  checked  by 
the  use  of  sulfurous  acid  and  good  fermentations  assured  by  the  use 
of  pure  selected  yeast. 


PLATE  1 

Apparatus  used  in  isolating  micro-organisms  from  grapes:  Mason  jar 
plugged  with  cotton  and  sterilized.  Sterile  petri  dishes.  Sterile  1  c.c.  pipettes 
Sterile  agar  and  sterile  must  in  tubes. 


[64] 


PLATE  2 

Colonies  of  yeasts  70,  71,  and  78,  on  agar  agar  and  gelatin.  Upper  row 
of  plates  agar  agar;  lower,  gelatin.  Reading  from  left  to  right  colonies  repre- 
sent numbers  70,  71,  and  78. 


[66] 


